Method for reprogramming cells

Abstract

The present disclosure provides a method for reprogramming cells in the presence of one or more reprogramming factors, reprogrammed cells and uses thereof, and kits comprising the reprogramming factors.

Description

[0001] This application is a divisional application of a Chinese patent application with an application number of 202011345680.2, a filing date of November 26, 2020, and an invention title of "Method for Reprogramming Cells". Technical Field

[0002] The present disclosure generally relates to methods for reprogramming cells. Background Art

[0003] Normally functioning corneal endothelial cells (CECs) maintain the transparency and proper fluid levels of the cornea, for example, maintaining a balance between fluid "leakage" into the stroma and continuous active pumping from the stroma to move fluid into the anterior chamber of the eye.

[0004] It has been reported that corneal endothelial cells have little or no in vivo proliferative capacity and thus cannot be naturally replaced when damaged or otherwise lost. In the human body, the corneal endothelial cell layer is most densely populated at birth, and the cell density rapidly decreases as the eye grows thereafter (such that the same number of cells cover a larger area). Thereafter, the corneal cell density gradually decreases with age, significantly reflecting the gradual loss of cells that are not replaced. As the cell density decreases, each cell spreads out and covers a larger area to maintain the barrier and pump functions of the cell layer. However, once the cell density drops too low (below about 500 to 1000 cells / mm²), its function is impaired, causing corneal opacity, stromal edema, loss of visual acuity, and ultimately blindness.

[0005] Although different treatment methods have been developed, there is a great need for new corneal endothelial reconstruction techniques. Summary of the Invention

[0006] The present disclosure provides a method for reprogramming a first type of cell into a second type of cell, which includes culturing the first type of cell in the presence of a first set of reprogramming factors, wherein the first set of reprogramming factors includes a glycogen synthase kinase 3 (GSK3) inhibitor, a transforming growth factor-β (TGFβ) inhibitor, and a cyclic AMP inducer.

[0007] In certain embodiments, the first set of reprogramming factors further includes basic fibroblast growth factor (bFGF), a DNA methyltransferase inhibitor, a histone methyltransferase inhibitor (e.g., a DOT1L inhibitor), a histone deacetylase (HDAC) inhibitor, BMP4, or a combination thereof.

[0008] In certain embodiments, the first set of reprogramming factors consists of: (a) a GSK3 inhibitor, a TGFβ inhibitor, and a cyclic AMP inducer, (b) a GSK3 inhibitor, a TGFβ inhibitor, a cyclic AMP inducer, and bFGF, or (c) a GSK3 inhibitor, a TGFβ inhibitor, a cyclic AMP inducer, a DNA methyltransferase inhibitor, a histone methyltransferase inhibitor (e.g., a DOT1L inhibitor), and a histone deacetylase inhibitor.

[0009] In certain embodiments, the GSK3 inhibitor is selected from the group consisting of: CHIR99021, LiCl, Li2CO3, and BIO ((2’Z,3’E)-6-bromoindirubin-3’-oxime), TD114-2, Kenpaullone, TWS119, CBM1078, SB216763, 3F8 (TOCRIS), AR-A014418, FRATide, indirubin 3’-oxime, and L803.

[0010] In certain embodiments, the TGFβ inhibitor is selected from the group consisting of: SB431542, Repsox, 616452, LDN193189, A8301, GW788388, SD208, SB525334, LY364947, D4476, SB505124, and Tranilast.

[0011] In certain embodiments, the cyclic AMP inducer is forskolin, IBMX, Rolipram, 8BrcAMP, prostaglandin E2 (PGE2), NKH477, dibutyryl-cyclic AMP (DBcAMP), Sp-8-BrcAMPs.

[0012] In certain embodiments, the DNA methyltransferase inhibitor is selected from the group consisting of: 5-aza-dC, Azacytidine, and RG108.

[0013] In certain embodiments, the DOT1L inhibitor is EPZ004777.

[0014] In certain embodiments, the histone deacetylase inhibitor is selected from the group consisting of: valproic acid (VPA), trichostatin A (TSA), vorinostat, depsipeptide, Trapoxin, Depudecin, FR901228, and butyrate.

[0015] In certain embodiments, the first type of cell is a somatic cell. In certain embodiments, the somatic cell is derived from the mesoderm, ectoderm, or endoderm.

[0016] In certain embodiments, the somatic cell is a fibroblast. In certain embodiments, the fibroblast is selected from the group consisting of mouse embryonic fibroblast (MEF), mouse tail tip fibroblast (TTF), human embryonic fibroblast (HEF), human neonatal fibroblast (HNF), human adult fibroblast (HAF), human foreskin fibroblast (HFF), and mixtures thereof.

[0017] In certain embodiments, the somatic cell is a human exfoliated renal epithelial cell.

[0018] In certain embodiments, the first type of cell is a stem cell. In certain embodiments, the stem cell is selected from the group consisting of human umbilical cord mesenchymal stem cell, human embryonic stem cell, and induced pluripotent stem cell (iPSC).

[0019] In certain embodiments, the second type of cell is a stem cell. In certain embodiments, the stem cell is a neural crest cell-like cell (NCC-like cell). In certain embodiments, the NCC-like cell is positive for P75, Hnk1, AP2α, and Sox10.

[0020] In certain embodiments, the first type of cell is cultured in the presence of a first set of reprogramming factors (a) for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days or (b) for no more than 20, 19, 18, 17, 16, 15, 14, 13, or 12 days.

[0021] The present disclosure provides a method of reprogramming a second type of cell into a third type of cell, which includes culturing the second type of cell in the presence of a second set of reprogramming factors, wherein the second set of reprogramming factors includes a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0022] In certain embodiments, the second set of reprogramming factors further includes BMP4 and / or a DNA methyltransferase inhibitor. In certain embodiments, the casein kinase 1 inhibitor is CKI-7. In certain embodiments, the DNA methyltransferase inhibitor is selected from the group consisting of 5-azacytidine, 5-aza-dC, and RG108.

[0023] In certain embodiments, the third type of cell is a somatic cell. In certain embodiments, the somatic cell is a corneal endothelial cell (CEC)-like cell (CEC-like cell). In certain embodiments, the CEC-like cell is positive for ZO-1 and Na + / K + -ATPase.

[0024] In certain embodiments, the second type of cells is cultured in the presence of the second set of reprogramming factors for (a) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days or (b) not more than 20, 19, 18, 17, 16, 15, 14, 13, or 12 days.

[0025] The present disclosure provides a method of reprogramming a first type of cells into a third type of cells, comprising the steps of (a) culturing the first type of cells in the presence of a first set of reprogramming factors, wherein the first set of reprogramming factors comprises a glycogen synthase kinase 3 (GSK3) inhibitor, a transforming growth factor (TGFβ) inhibitor, and a cyclic AMP inducer, and (b) culturing the cells obtained from step (a) in the presence of a second set of reprogramming factors, wherein the second set of reprogramming factors comprises a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0026] In certain embodiments, the method further comprises washing the cells obtained from step (a) before starting step (b). In certain embodiments, there is no washing step between step (a) and step (b).

[0027] The present disclosure provides a method of reprogramming a first type of cells into a third type of cells, which comprises culturing the first type of cells in the presence of

[0028] a first set of reprogramming factors and a second set of reprogramming factors, wherein the first set of reprogramming factors comprises a glycogen synthase kinase 3 (GSK3) inhibitor, a transforming growth factor (TGFβ) inhibitor, and a cyclic AMP inducer, and the second set of reprogramming factors comprises a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0029] The present disclosure provides a population of NCC-like cells produced according to the methods provided herein.

[0030] The present disclosure provides a population of corneal endothelial cell-like cells (CEC-like cells) produced according to the methods provided herein.

[0031] The present disclosure provides a composition comprising the NCC-like cells or CEC-like cells provided herein.

[0032] The present disclosure provides a method of treating a disease or condition associated with dysfunctional or damaged corneal endothelial cells, which comprises administering to a subject in need thereof an effective amount of the CEC-like cells provided herein or the composition provided herein. In certain embodiments, the subject is a human.

[0033] In certain embodiments, the disease or medical condition is selected from the group consisting of: Fuch's dystrophy, iridocorneal endothelial syndrome, posterior polymorphous dystrophy, congenital hereditary endothelial dystrophy, age-related macular degeneration (AMD), retinitis pigmentosa, glaucoma, corneal dystrophy, contact lens use, late endothelial failure in cataract surgery, and corneal transplantation.

[0034] The present disclosure provides a kit for reprogramming a first type of cell into a second type of cell, wherein the kit comprises a first set of reprogramming factors, and the first set of reprogramming factors comprises a glycogen synthase kinase 3 (GSK3) inhibitor, a TGFβ inhibitor, and a cyclic AMP inducer.

[0035] The present disclosure provides a kit for reprogramming a second type of cell into a third type of cell, wherein the kit comprises a second set of reprogramming factors, and the second set of reprogramming factors comprises a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0036] The present disclosure provides a kit for reprogramming a first type of cell into a third type of cell, wherein the kit comprises the first set of reprogramming factors and the second set of reprogramming factors, wherein the first set of reprogramming factors comprises a glycogen synthase kinase 3 (GSK3) inhibitor, a TGFβ inhibitor, and a cyclic AMP inducer, and the second set of reprogramming factors comprises a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0037] The present disclosure provides a method for identifying a drug that affects the effect of NCC, chemically induced NCC (ciNCC), or chemically induced CEC (ciCEC), which comprises administering a drug candidate to NCC, ciNCC, or ciCEC, and detecting the response of the cells to the drug candidate, thereby identifying the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Shown is the conversion of fibroblasts into ciNCC by conditions determined by chemical composition. A) Schematic illustration of the induction method starting from mouse fibroblasts. B) Shows morphological changes at different time points during neural crest cell-like cell induction. C) Shows the number of ciNCC colonies generated under specified conditions. Data are mean ± SD, n = 3 independent experiments. D) Shows an image of SOX10+ colonies taken in situ by fluorescence microscopy. E) Shows the number of SOX10+ colonies generated under specified conditions. Data are mean ± SD, n = 3 independent experiments. F) Shows immunostaining of ciNCC markers P75, Hnk1, and AP2α at day 12.

[0039] Figure 2Show the differentiation potential of ciNCCs transformed from fibroblasts. A) Show immunocytochemical analysis, which shows that ciNCCs can differentiate into peripheral neurons (as indicated by immunocytochemistry of Tuj1 and peripherin), and can also differentiate into Schwann cells (as indicated by immunocytochemistry of S100β and GFAP). The bright-field image on the right shows melanocytes differentiated from ciNCCs. B) Show that ciNCCs can further differentiate into chondrocytes, adipocytes, and osteocytes (as shown by Alcian blue staining, Oil Red O staining, and Alizarin Red staining, respectively).

[0040] Figure 3 Show that ciNCCs can further differentiate into corneal endothelial cell-like cells (or chemically induced CECs, ciCECs). Figure 3 A shows immunofluorescence staining of corneal endothelial markers (including Na + -K + ATPase, AQP1, Vimentin, N-cadherin, laminin, and AQP1). Nuclei were stained with DAPI. Scale bar, 50 μm. Figure 3 B shows transmission electron microscopy images, which show tight junctions of corneal endothelial cells. Figure 3 C shows the protocol of a two-step lineage reprogramming strategy for functionally generating corneal endothelial cells from fibroblasts.

[0041] Figure 4 Show lineage tracing of fibroblast reprogramming towards corneal endothelial cell-like cells (or ciCECs). A) Show a schematic diagram of a genetic fate mapping method for tracing the origin of corneal endothelial cell-like cells reprogrammed from Fsp1-Cre:R26R tdTomato MEFs. B) Show lineage tracing of the origin of corneal endothelial cell-like cells reprogrammed from Fsp1-Cre:R26R tdTomato MEFs. C) Show immunocytochemical analysis, which shows that FSP1-ciNCCs are positive for P75, Hnk1, AP2α, and SOX10. D) Show representative morphological changes of these MEF-induced Fsp1-Cre:R26R tdTomato MEFs and ciCECs.

[0042] Figure 5Demonstration of the generation of corneal endothelial cell-like cells from different human cell types by small molecules. Human embryonic skin fibroblasts (HEF), human neonatal fibroblasts (HNF), adult fibroblasts (HAF), human umbilical cord mesenchymal stromal cells (MSC), and urine cells (UC).

[0043] Figure 6 Demonstration of the clinical observation results of the ciCEC group and the control group in rabbits at different days. A) Bright-field images of ciCEC at passage 10 (P10) are shown. ciCEC at passage 10 expresses Na + -K + ATPase and ZO-1. B) Slit-lamp photographs are shown, which show that the transparency of the cornea in the ciCEC group was significantly improved after injection on day 7 of ciCEC transplantation (Figure 1), while corneal opacity and stromal edema remained severe in the untreated control group analyzed by slit-lamp photographs (Figure 3). C) Demonstration of the significant corneal thickness differences between the CEC-like cell group and the control group analyzed by Visante OCT. D) Confocal microscope images are shown, which confirm the full coverage of polygonal cells on Descemet's membrane in the ciCEC group. E) Demonstration of the corneal thickness changes during clinical observation in the low-dose (1×10 6 cells / ml ciCEC), high-dose (2×10 6 cells / ml ciCEC), and PBS-treated control groups. B)-D) Images from left to right: Image 1: ciCEC-treated group, Image 2: intact contralateral eye, Image 3: PBS-treated control group, Image 4: normal eye group.

[0044] Figure 7 Slit-lamp photographs of the ciCEC group and the control group in rabbits at different days are shown. The slit-lamp photographs show that the transparency of the cornea in the ciCEC group was significantly improved after injection, and the pupil and iris texture could be seen (ciCEC group, upper figure). Only about 14 days later, the cornea became significantly transparent, while corneal opacity and stromal edema remained severe in the control group (control group, upper figure). The lower figures of both the ciCEC group and the control group show the corneal reflex detected by slit-lamp, and each image in the lower figures corresponds to the image in its upper figure.

[0045] Figure 8Showing the conversion of MEFs into ciNCCs by small molecule induction. A) Schematic diagram of reprogramming NCCs from MEFs. B) Effects of individual chemicals on ciNCC generation. Data are mean ± SD, n = 3 independent experiments. C) Promoting effects of individual chemicals on ciNCC generation (data are mean ± SD, n = 3 independent experiments). D) Schematic illustration of our strategy for converting MEFs into ciNCCs. E) Generation of Wnt1 + ciNCCs from MEFs using a small molecule cocktail. F) Quantification of the number of Wnt1+ cells in ciNCC clusters induced by candidate cocktails in a combinatorial screen (independent experiments, n = 3). G) Morphological changes at different days during ciNCC induction treatment (scale bar, 50 μm). H) Percentage of Wnt1 - tdTomato + cells at different days (independent experiments, n = 3).

[0046] Figure 9 Showing the characterization of M6-induced ciNCCs. A) Morphology of M6-induced ciNCCs (scale bar, 400 μm). B) Immunostaining showing that MEF-derived ciNCCs express P75, HNK1, AP2a, and Nestin (scale bar, 50 μm). C) Representative images of differentiated ciNCCs stained with peripheral neuron markers (scale bar, 50 μm). D) Differentiation of ciNCCs into Schwann cells and melanocytes and marker expression (scale bar, 50 μm). E) Differentiation of ciNCCs into mesenchymal lineages and further differentiation into adipocytes, chondrocytes, and osteocytes (scale bar, 100 μm).

[0047] Figure 10 Showing the generation of murine ciCECs from fibroblasts by small molecule induction. A) Schematic diagram of chemically reprogramming ciCECs from MEFs. B) Bright-field images of initial MEFs, reprogrammed ciNCC colonies, and ciCECs (scale bar, 400 μm). C) Morphological changes at different days during the induction of ciCECs from Wnt1 - tdTomato + ciNCCs (scale bar, 400 μm). D) ciCECs were stained for the corneal endothelial marker Na + / K +Immunofluorescence staining of -ATPase, AQP1, vimentin, N-cadherin, laminin, and ZO-1 (scale bar, 50 μm). E) LDL uptake function in ciCEC (scale bar, 50 μm). F) Heatmap of differentially expressed genes in samples at designated time points. These numbers below the heatmap indicate independent biological replicates. Red and blue indicate upregulated and downregulated genes, respectively. G) TEM of ciCEC showing tight junctions (scale bar, 5 μm).

[0048] Figure 11 Show gene expression profiling of ciNCC and ciCEC. A) qRT-PCR analysis showing NC gene expression at designated time points. Gene expression (log2) was normalized relative to gene expression in MEF. B) qRT-PCR analysis of designated NC cell gene expression in ciCEC, MEF, and pCEC derived from MEF at different passages. C) Heatmap of differentially expressed genes in samples at designated time points. The numbers below the heatmap indicate independent biological replicates (n = 2 - 3). Red and blue indicate upregulated and downregulated genes, respectively. D) Principal component analysis of samples from day 0 (D0), day 7 (D7), and day 12 (D12) of reprogramming, ciCEC, and control pCEC.

[0049] Figure 12 Show lineage tracing to confirm the induction of ciCEC from fibroblasts. A) Schematic diagram showing the genetic lineage tracing strategy. MEF was obtained by sorting p75 tdTomato / tdTomato - cells from MEF of E13.5 mouse embryos with Fsp1-Cre / ROSA26 + background. B) Show the FACS sorting results of p75 tdTomato / tdTomato - cells from MEF with Fsp1-Cre / ROSA26 + genetic background. C) Show the immunostaining analysis negative for Sox10, P75, Olig2, Hnk1, AP2, Sox2, and Pax6 in p75 - / tdTomato + cells (scale bar, 100 μm). D) Differentiation of p75 - / tdTomato + cells towards ciNCC and ciCEC (scale bar, 400 μm). e) Show the immunostaining analysis positive for ZO-1, laminin, Na + / K + -ATPase, and AQP1 in ciCEC derived from Fsp1-tdTomato-MEF (scale bar, 50 μm).

[0050] Figure 13 Show that small molecules promote long-term expansion of ciCECs. A) ciCECs were expanded in serum-free control medium for 3 days (scale bar, 200 μm). B) Continuous expansion of ciCECs in serum-free medium supplemented with SB431542 and CKI-7 (scale bar, 200 μm). C) The average population doubling time of ciCECs cultured in medium with or without SB431542 and CK1-7 (mean ± SD, n = 3; ***p < 0.001). D) Bright-field images of ciCECs at P5, which were expanded for 3 days under culture conditions supplemented with SB431542 and CKI-7 (scale bar, 50 μm). E) These ciCECs at P30 were fixed and stained for Na + / K + -ATPase, AQP1, and ZO-1 (scale bar, 50 μm).

[0051] Figure 14 Show in vivo transplantation of ciCECs. A) Diagram depicting the transplantation of ciCECs and ROCK inhibitor into a model rabbit. B) After transplantation, corneal transparency in the transplanted eye was significantly improved, while corneal opacity and stromal edema remained severe in the untreated control. C) Slit-lamp microscopic images showing that after transplantation, corneal transparency in the transplanted cornea was significantly improved, while corneal opacity and stromal edema still existed in the untreated control. D) Show immunohistochemistry of viable tdTomato + ciCECs attached to Descemet's membrane (scale bar, 100 μm). E) Visante OCT showing improved corneal edema (reduced corneal thickness) in the transplanted eye. F) Trend of corneal thickness after transplantation. There was a significant difference in corneal thickness between the untreated control and the transplanted control. Results are the mean and SEM of biological replicates (n = 9). G) Real-time confocal imaging of corneal endothelium, which confirmed the complete coverage of polygonal cells on Descemet's membrane in the transplanted eye.

[0052] Figure 15 Show the characterization of Wnt1 - MEFs. A) Schematic diagram showing the genetic lineage tracing strategy. Wnt1 - MEFs were obtained by sorting tdTomato - cells from MEFs derived from E13.5 mouse embryos of the Wnt1 - Cre / ROSA26tdTomato background. B) Show the FACS sorting results of tdTomato - cells from MEFs. C) Show Wnt1 -Immunostaining analysis of negative results for P75, HNK1, Sox10, and Ap2 in MEFs (scale bar, 100 μm). D) Shows Wnt1 - RT-PCR analysis of specified neural crest gene expression in MEFs and primary NCCs. Gapdh served as a control. E) Shows Wnt1 - Representative image of MEF-derived ciNCC colonies (scale bar, 400 μm).

[0053] Figure 16 Shows that M6 converts tdMEFs into ciNCCs. A) Morphology of tdMEFs and tdMEF-derived ciNCCs (scale bar, 400 μm). B) Representative image of ciNCCs differentiating into ciCECs (scale bar, 400 μm). C) Immunostaining analysis showing tdMEF-derived ciNCCs expressing NC cell markers (P75, HNK1, Sox10, and Ap2α) (scale bar, 50 μm). D) FSP1-tdTomato + Differentiation process of ciCECs.

[0054] Figure 17 Shows generation of ciCECs from fibroblasts by small molecules without passing through the iPSC stage. A) Morphological changes at different time points during induction of ciCECs from MEFs (scale bar, 400 μm). B) Immunofluorescent staining of MEF-derived ciNCC colonies for the NC marker Sox10 (scale bar, 400 μm). C) Morphological changes at different time points during induction of ciCECs from OG-MEFs (scale bar, 400 μm). D) By FACS analysis, there were no Oct4-GFP positive cells for ciCEC induction. E) Typical ciCEC karyotype (passage 10).

[0055] Figure 18 Shows the proliferative potential of ciCECs. A) Representative images of ciCECs at P3 and pCECs at P3 (scale bar, 400 μm). B) Immunofluorescent images of Ki67 and ZO-1 expression in ciCECs at P3 and pCECs at P3. C) EdU incorporation experiments of ciCECs and pCECs at different passage numbers analyzed by flow cytometry. D) Distribution of ciCECs and pCECs in the cell cycle (G1, S, and G2 phases) (scale bar, 400 μm). Scratch ciCECs at P3 and pCECs at P3, and migration was observed and images were taken after 8 hours and 20 hours. E) Scratch wound assay of pCECs at P3 and ciCECs at P3, P15, and P30 (scale bar, 200 μm). Migration was observed after 8 hours and 20 hours. F) Shows quantification of scratch closure.

[0056] Figure 19 Show the observation results of the transplanted eyes in the rabbit model. A) Slit lamp microscopy (middle) and confocal microscopy images (right) of the transplanted eyes in Subject #10 obtained at different days at baseline (before cell injection) and after ciCEC injection supplemented with ROCK inhibitor. B) Visante OCT showing corneal thickness in the eyes transplanted with ciCEC at different days. C) Show viable tdTomato connected to Descemet's membrane + Immunostaining analysis of ciCEC. D) Changes in corneal thickness at different days. Detailed implementation mode

[0057] The following description of the present disclosure is only intended to illustrate various embodiments of the present disclosure. Therefore, the specific modifications discussed should not be construed as limitations on the scope of the present disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the present disclosure, and it should be understood that such equivalent embodiments will be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0058] A. General Definitions

[0059] Unless the context clearly indicates otherwise, the singular terms "a / an" and "the" include plural referents. By way of example, reference to "a cell" refers to one or more cells, and reference to "the method" includes reference to equivalent steps and methods disclosed herein and / or known to those skilled in the art, and so on. Similarly, unless the context clearly indicates otherwise, the word "or" is intended to include "and". Although methods and materials similar or equivalent to those described herein can be used to practice or test the present disclosure, suitable methods and materials are described below. The abbreviation "e.g." is derived from the Latin exempli gratia and is used herein to indicate non-limiting examples. Thus, the abbreviation "e.g." is synonymous with the term "for example".

[0060] As used herein, the term "comprising / comprises" is used when referring to a composition, a method, and its corresponding components essential for the method or composition, but is still open for including unspecified elements, whether necessary or not.

[0061] The term "consisting of" refers to a composition, a method, and its corresponding components as described herein, which excludes any element not recited in the description of the embodiment.

[0062] The term "about" or "substantially" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

[0063] As used herein, the term "cell" refers to a single cell, a cell line, or a culture derived from such cells.

[0064] As used herein, the term "reprogram / reprogramming / reprogrammed" or its equivalent refers to the process of altering or reversing the differentiation state of a cell in culture or in vivo as compared to the differentiation state the cell would have under the same conditions without reprogramming. In other words, in the context of the present disclosure, "reprogramming" includes differentiation, dedifferentiation, and transdifferentiation. As used herein, the term "differentiation" refers to a cellular process by which less specialized cells become more specialized cell types. In contrast, the term "dedifferentiation" refers to a cellular process in which partially or terminally differentiated cells revert to an earlier developmental stage, such as a pluripotent or multipotent cell. Further in contrast, the term "transdifferentiation" refers to a cellular process of converting one differentiated cell type into another differentiated cell type. Thus, as used herein, the terms "less differentiated state" or "less specialized" or "earlier developmental stage" are relative terms and include a fully dedifferentiated state (or complete dedifferentiation) and a partially differentiated state (or partial differentiation). To distinguish from the above cell development, an "undifferentiated cell" is a cell that can differentiate in many directions, i.e., it can differentiate into two or more types of specialized cells. A typical example of an undifferentiated cell is a stem cell.

[0065] Cell types pass through various levels of potency during differentiation, such as totipotency, pluripotency, and multipotency. The phrase "totipotent stem cell" refers to a cell that can differentiate into all of the cells that make up an organism, such as the cell produced by the fusion of an egg cell and a sperm cell. Cells produced by the first few divisions of a fertilized egg may also be totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells, such as ES cells, can give rise to any fetal and adult cell type. However, they cannot develop into a fetus or adult animal alone because they lack the potential to develop extraembryonic tissues. Extraembryonic tissues are partly derived from the extraembryonic endoderm and can be further classified into parietal endoderm (Reichert's membrane) and visceral endoderm (the part that forms the yolk sac). Both the parietal endoderm and the visceral endoderm support embryonic development but do not themselves form embryonic structures. There are also other extraembryonic tissues, including extraembryonic mesoderm and extraembryonic ectoderm. As used herein, "pluripotent stem cells" or cells with "pluripotency" or equivalents refer to a population of cells that can differentiate into all three germ layers (e.g., endoderm, mesoderm, and ectoderm). Pluripotent cells express a variety of pluripotent cell-specific markers, have the morphological characteristics of undifferentiated cells (i.e., compact colonies, high nucleus-to-cytoplasm ratio, and prominent nucleoli), and form teratomas when introduced into immunocompromised animals such as SCID mice. Teratomas typically contain cells or tissue characteristics of all three germ layers. One of ordinary skill in the art can assess these characteristics by using techniques commonly used in the art. See, e.g., Thomson et al., Science 282:1145-1147 (1998). Pluripotent cells are capable of proliferating in cell culture and differentiating towards multiple lineage-restricted cell populations that exhibit multipotent properties. Multipotent stem cells or cells with multipotency or equivalents are more differentiated than pluripotent stem cells but are not terminally differentiated. Pluripotent stem cells thus have a higher potential than multipotent stem cells.

[0066] B. Reprogrammed Cells

[0067] In one aspect, the present disclosure provides a method for reprogramming a first type of cell into a second type of cell, comprising culturing the first type of cell in the presence of a first set of reprogramming factors, wherein the first set of reprogramming factors comprises a glycogen synthase kinase 3 (GSK3) inhibitor, a transforming growth factor-β (TGFβ) inhibitor, and a cyclic AMP inducer.

[0068] In some embodiments, culturing a first type of cell in the presence of a first set of reprogramming factors causes at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the cells to become a second type of cell.

[0069] In certain embodiments, the first type of cell is a mammalian cell derived from the mesoderm, ectoderm, or endoderm, such as a human or mouse cell.

[0070] The ectoderm, mesoderm, and endoderm are the three germ layers that form during embryonic development, where the mesoderm is the middle layer, the ectoderm is the outer layer, and the endoderm is the inner layer. The mesoderm forms mesenchyme, mesothelium, non-epithelial blood cells, and coelomic cells, which make up muscle (smooth and striated), bone, cartilage, connective tissue, adipose tissue, the circulatory system, the lymphatic system, the dermis, the urogenital system, the serosa, and the notochord. The endoderm forms the pharynx, esophagus, stomach, small intestine, colon, liver, pancreas, bladder, the epithelial portions of the trachea and bronchi, the lungs, the thyroid, and the parathyroid glands. The ectoderm forms the surface ectoderm, neural crest, and neural tube, where the surface ectoderm develops into the epidermis, hair, nails, the lens of the eye, sebaceous glands, the cornea, dental enamel, and the epithelium of the mouth and nose; the neural crest of the ectoderm develops into the peripheral nervous system, the adrenal medulla, melanocytes, facial cartilage, and dentin; and the neural tube of the ectoderm develops into the brain, spinal cord, posterior pituitary, motor neurons, and the retina.

[0071] In certain embodiments, the first type of cell is a somatic cell. As used herein, the term "somatic cell" refers to any cell other than germ line cells (e.g., sperm and eggs, and the cells that make them (gametocytes)) and undifferentiated stem cells. The viscera, skin, bone, blood, and connective tissue are all composed of somatic cells. Somatic cells can be any type of somatic cell of any origin. By way of example, somatic cells can include, but are not limited to, fibroblasts, epithelial cells, supporting cells, endothelial cells, epithelial granular layer, neurons, islet cells, epidermal cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), red blood cells, macrophages, monocytes, mononuclear cells, cardiomyocytes, and other muscle cells.

[0072] In certain embodiments, the first type of cell is a mesodermal cell. Markers of mesodermal cells are known in the art, such as CD56 and APJ. In certain embodiments, mesodermal cells include fibroblasts, epithelial cells, leukocytes, adipocytes, and keratinocytes. In certain embodiments, the first type of cell is a fibroblast, including but not limited to mouse embryonic fibroblasts (MEF), mouse neonatal fibroblasts (MNF), mouse tail tip fibroblasts (TTF), human embryonic skin fibroblasts (HEF), human neonatal fibroblasts (HNF), adult human fibroblasts (HAF), human embryonic lung fibroblasts, human foreskin fibroblasts (HFF), and mixtures thereof.

[0073] Fibroblasts can be obtained from any suitable source, such as from commercial sources or from various organ tissues or skin tissues. Preferred fibroblasts are lung fibroblasts, foreskin fibroblasts, and adult dermal fibroblasts. In certain embodiments, fibroblasts are obtained from a patient, for example, by skin biopsy (e.g., reprogramming of human somatic cells to pluripotency with defined factors. George Q. Daley et al., Nature 2008; a method for the isolation and serial propagation of keratinocytes, endothelial cells, and fibroblasts from a single punch biopsy of human skin, Normand et al., In Vitro Cellular & Developmental Biology - Animal, 1995).

[0074] In certain embodiments, the first type of cell is an epithelial cell, such as human exfoliated renal epithelial cells. Epithelial cells can be obtained from urine samples.

[0075] In certain embodiments, the first type of cell is a leucocyte. The leucocytes can be obtained from a blood sample. Leucocytes are white blood cells that can generally be differentially classified into their subsets (lymphocytes, monocytes, neutrophils, eosinophils, and basophils) by: RF signal intensity (change in impedance at high frequency), DC signal intensity (change in direct current caused by differences in conductivity between suspended particles and the liquid medium in which the particles are suspended), fluorescence intensity, scattered light intensity, absorbance, depolarization of scattered light, etc. (see US5618733A).

[0076] In certain embodiments, the first type of cell is an adipocyte or a keratinocyte. Adipocytes and keratinocytes can also be readily obtained by skin biopsy or plucked hair (isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells, Belmonte et al., Nature Protocols 2010).

[0077] In other embodiments, the first type of cell is a somatic cell. As used herein, the term "somatic cell" refers to cells found throughout the body after embryonic development. In certain embodiments, the first type of cell can be a stem cell, such as an embryonic stem cell, an induced pluripotent stem cell (iPSC), and an adult stem cell, including but not limited to hematopoietic stem cells, vascular endothelial stem cells, cardiac stem cells, myogenic stem cells, mesenchymal stem cells, epidermal stem cells, adipose-derived stem cells, intestinal stem cells, neural stem cells, renal epithelial stem cells, urethral epithelial stem cells, and hepatic stem cells.

[0078] In some embodiments, a stem cell refers to an undifferentiated cell that is capable of proliferating and giving rise to more progenitor cells, which can generate a large number of blast cells, which in turn can produce differentiated or differentiable daughter cells. Stem cells can divide asymmetrically, where one daughter cell retains the stem cell state and the other daughter cell expresses some unique other specific functions and phenotypes. Alternatively, some of the stem cells in a population can divide symmetrically into two stem cells, thus maintaining some stem cells in the population as a whole, while the other cells in the population only produce differentiated progeny. The daughter cells themselves can be induced to proliferate and produce progeny, which then differentiate into one or more mature cell types, while also retaining one or more cells with parental developmental potential. In other embodiments, the term "stem cell" refers to a subset of progenitor cells that have the ability or potential to differentiate into a more specialized or differentiated phenotype in a particular environment, and that retain the ability to proliferate without substantial differentiation in some cases. In one embodiment, the term stem cell generally refers to naturally occurring blast cells, the progeny (offspring) of which typically specialize in different directions through differentiation (e.g., by acquiring fully individual characteristics), as occurs in the progressive diversification of embryonic cells and tissues. Differentiated cells can be derived from pluripotent cells, which themselves are derived from pluripotent cells, and so on. Although each of these pluripotent cells can be considered a stem cell, the range of cell types that each can produce can vary significantly. In many biological instances, stem cells are also "pluripotent" because they can produce offspring of more than one unique cell type, but this is not required for "stem-ness". Self-renewal is another typical part of the definition of a stem cell. In theory, self-renewal can occur through either of two major mechanisms.

[0079] The term "embryonic stem cell" is used to refer to the pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see U.S. Patent Nos. 5,843,780 and 6,200,806, which are incorporated herein by reference). The distinguishing characteristics of embryonic stem cells define the embryonic stem cell phenotype. Thus, if a cell has one or more of the unique characteristics of embryonic stem cells that enable the cell to be distinguished from other cells, then the cell has the embryonic stem cell phenotype. Exemplary distinguishing characteristics of embryonic stem cells include, but are not limited to, gene expression profiles, proliferative capacity, differentiation capacity, karyotype, responsiveness to specific culture conditions, and the like.

[0080] The term "adult stem cell" or "ASC" is used to refer to any multipotent stem cell derived from non-embryonic tissues (including fetal, juvenile, and adult tissues). Adult stem cells have been isolated from a wide variety of adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stem cells can be characterized based on gene expression, factor responsiveness, and morphology in culture. As shown above, stem cells have been found to reside in almost every tissue. Thus, it can be appreciated from the techniques described herein that stem cell populations can be isolated from almost any animal tissue.

[0081] In certain embodiments, the first type of cell can be a mesenchymal stem cell, a mesenchymal stromal cell, a human embryonic stem cell, or an induced pluripotent stem cell (iPSC).

[0082] As used herein, the terms "iPS cell", "iPSC", and "induced pluripotent stem cell" are used interchangeably and refer to a pluripotent cell that is artificially derived (e.g., by complete or partial reprogramming induction) from a differentiated somatic cell (i.e., derived from a non-pluripotent cell). A pluripotent cell can differentiate into all cells of the three developmental germ layers.

[0083] Mesenchymal stem cells (MSCs) or mesenchymal stromal cells are adult stem cells that are traditionally found in bone marrow. However, mesenchymal stem cells can also be isolated from other tissues, including umbilical cord blood, peripheral blood, fallopian tubes, and fetal liver and lung. MSCs can differentiate into a variety of cell types, including osteoblasts (bone cells), chondrocytes (chondrocyte / cartilage cell), myocytes (muscle cells), and adipocytes (adipocyte) (fat cells that produce bone marrow adipose tissue (fat cell)).

[0084] In certain embodiments, the first type of cell is an ectodermal cell. In certain embodiments, the first type of cell is an endodermal cell.

[0085] In certain embodiments, the second type of cell transdifferentiates from the first type of cell. For example, mesodermal cells are reprogrammed into ectodermal cells. In certain embodiments, the second type of cell is an ectodermal cell. In certain embodiments, the multipotent stem cell is a neural crest cell (NCC) or a neural crest cell-like cell. In certain embodiments, the second type of cell is a neural crest cell (NCC) or a neural crest cell-like cell.

[0086] "Neural crest cells" or "NCCs" generally refer to neural progenitor cells that have the developmental potential to give rise to pigment cells that co-express melanosome markers and HMB45. Neural crest cells can be identified by expressing markers identified herein and known in the art. To distinguish the chemically induced NCCs (ciNCCs) provided herein from primary NCCs, the resulting ciNCCs are also named neural crest cell-like cells (NCC-like cells). In some embodiments, the resulting NCC-like cells of NCCs exhibit one or more biomarkers consistent with the primary NCC phenotype. In some embodiments, the resulting NCC-like cells lack the expression or have lower expression of one or more biomarkers consistent with the NCC phenotype (e.g., the expression of PAX6). Exemplary NC biomarkers that are present and consistent with the NCC phenotype can include Nestin, SOX10, SOX9, HNK-1, P75 (NGFR), AP2α, PAX3, PAX7, SNAI2, Snail, Twistl, Krox20, CD271, FoxD3, AN2, and Ki67, and / or at least one pluripotency marker NANOG, ZNF206, or OCT4. In some embodiments, the NC biomarker is P75, Hnk1, AP2α, and / or SOX10. In an embodiment, the expression of the NC biomarker is increased by more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000-fold or more relative to the first type of cell from which the NCC-like cells are generated.

[0087] In certain embodiments, the first type of cell is a fibroblast and the second type of cell is an NCC-like cell. In certain embodiments, the first type of cell is a mouse embryonic fibroblast (MEF) and the second type of cell is an NCC-like cell. In certain embodiments, the first type of cell is a mouse tail tip fibroblast (TTF) and the second type of cell is an NCC-like cell. In certain embodiments, the first type of cell is a human embryonic skin fibroblast (HEF) and the second type of cell is an NCC-like cell. In certain embodiments, the first type of cell is a human embryonic lung fibroblast, a human foreskin fibroblast (HFF), and the second type of cell is an NCC-like cell. In certain embodiments, the first type of cell is a human neonatal fibroblast (HNF) or an adult fibroblast (HAF), and the second type of cell is an NCC-like cell.

[0088] In certain embodiments, the first type of cell is an epithelial cell (e.g., human exfoliated renal epithelial cells) and the second type of cell is an NCC-like cell. In certain embodiments, the first type of cell is a white blood cell (e.g., lymphocyte, monocyte, neutrophil, eosinophil, and basophil) and the second type of cell is an NCC-like cell. In certain embodiments, the first type of cell is an adipocyte and the second type of cell is an NCC-like cell. In certain embodiments, the first type of cell is a keratinocyte and the second type of cell is an NCC-like cell. In certain embodiments, the first type of cell is an MSC, human embryonic stem (ES) cell, or iPSC and the second type of cell is an NCC-like cell.

[0089] In certain embodiments, the second type of cell is certain types of pluripotent stem cells reprogrammed from another type of stem cell (the first type of cell) (such as mesenchymal stem cells, human embryonic stem cells, or induced pluripotent stem cells (iPSCs)).

[0090] Expression of the marker can be detected by any method known in the art, including but not limited to Western blotting, mRNA amplification-based methods (e.g., PCR, isothermal amplification, etc., which may include reverse transcription and can be applied to detect expression from a single cell or multiple cells), Northern blotting, immunostaining, etc. Additionally, expression of the marker can be inferred by the expression of a reporter construct (such as a fluorescent protein whose expression can be visually detected, an antibiotic resistance gene whose expression can be detected by cell survival in the presence of an antibiotic, etc.), where the reporter construct is under the control of a gene element that confers cell type-specific expression (such as the promoter of one of the aforementioned markers or a fragment thereof). Exemplary reporter constructs are the pOCT4-GFP and pOCT4-LUC genes, which drive the expression of GFP and luciferase, respectively, in ES cells, and the expression of either GFP or luciferase can be easily detected using conventional methods. Other methods for detecting marker expression that can be used are known in the art. See generally Ausubel, Current Protocols in Molecular Biology (Current Protocols, 1988); Ausubel et al., Short Protocols in Molecular Biology (Current Protocols; 5th Edition, 2002); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 3rd Edition, 2001); Sambrook et al., The Condensed Protocols from Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2006), each of which is incorporated herein by reference in its entirety.

[0091] Any cell culture system known in the art can be used in the present disclosure. In certain embodiments, an adherent culture system is used in the methods of the present disclosure. The term "adherent culture" refers to a cell culture system in which cells are cultured on a solid surface, which can in turn be coated with a matrix. The cells may or may not adhere tightly to the solid surface or the matrix. The matrix for adherent culture may further comprise, for example, any one or combination of the following: polystyrene, polyester, polycarbonate, poly(N-isopropylacrylamide), polyornithine, laminin, polylysine, purified collagen, gelatin, cellulose, extracellular matrix, fibronectin, tenacin, vitronectin, polyglycolic acid (PGA), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), Matrigel, hydroxyapatite, and amniotic membrane.

[0092] In certain embodiments, suspension culture can be used in the methods of the present disclosure. As used herein, the term "suspension culture" refers to a cell culture mode in which cells do not adhere to a solid support or a culture vessel. To transfer cells into a suspension culture, the cells are removed from the culture vessel, for example, by a cell scraper and transferred to a sterile low-adhesion culture plate containing a culture medium, which does not allow the cells to adhere to the surface of the culture plate. Thus, the cells can be cultured in suspension without adhering to the matrix or the bottom of the culture dish.

[0093] A culture medium suitable for culturing cells is any medium suitable for growing a particular cell type in a culture dish. Such media include, for example, Ham’s F10 (Sigma), Ham’s F12 medium, Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma), IMDM medium, Medium 199, Eagle's Minimum Essential Medium (EMEM), aMEM medium, Fischer's medium, Neurobasal medium (Life Technologies Corporation), and mixtures of these media. Additionally, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Patent Reissue No. 30,985 may be used as the culture medium. Any of these media may be supplemented as needed with the required salts (such as sodium chloride, calcium salts, magnesium salts, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds that are typically present at final concentrations in the micromolar range), glucose or an equivalent energy source, albumin, insulin, transferrin, selenium, fatty acids, 2-mercaptoethanol, thioglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antioxidants, pyruvate, cytokines, and the like. Any other necessary supplements at appropriate concentrations known to those skilled in the art may also be included. Culture conditions (such as temperature, pH, etc.) are those previously used for cell culture and will be apparent to one of ordinary skill in the art.

[0094] In the cell reprogramming methods described herein, cells are cultured in a basal medium supplemented with one or more reprogramming factors described herein. For example, prior to addition of a first set of reprogramming factors, the basal medium can contain DMEM / F12 / Glutamax (GIBCO), 10% KnockOut Serum Replacement (KSR) (GIBCO), 1% non-essential amino acids (NEAA) (GIBCO), 10% fetal bovine serum (FBS) (GIBCO), and 0.1 mM 2-mercaptoethanol (GIBCO). In another embodiment, prior to addition of a first set of reprogramming factors, the basal medium can contain DMEM / F12 / Glutamax (GIBCO), 0.075% bovine serum albumin (BSA) (GIBCO), 1% non-essential amino acids (NEAA) (GIBCO), and 0.1 mM 2-mercaptoethanol (GIBCO). For another example, prior to addition of a second set of reprogramming factors, the basal medium can contain DMEM / F12 / Glutamax (GIBCO), 10% KnockOut Serum Replacement (KSR) (GIBCO), 1% non-essential amino acids (NEAA) (GIBCO), and 0.1 mM 2-mercaptoethanol (GIBCO). In certain embodiments, the basal medium for the second set of reprogramming factors is serum-free. Those skilled in the art will appreciate that other necessary supplements (as described) can be added to the medium.

[0095] For the culture temperature, it has been demonstrated that culturing at a temperature of 35.0 °C or higher promotes cell reprogramming. The culture temperature is a temperature that does not damage the cells, such as preferably 35.0 °C to 42.0 °C, or more preferably 36.0 °C to 40.0 °C, or more preferably 37.0 °C to 39.0 °C.

[0096] A reprogramming factor is a molecule that can cause reprogramming alone or in combination with other molecules when in contact with the cell (e.g., expressed by the cell, transformed into the cell for expression, provided exogenously to the cell, etc.). Reprogramming factors can be provided from exogenous sources, such as by addition to the medium, and can be introduced into the cell by methods known in the art, such as by coupling to a cell-penetrating peptide, protein or nucleic acid transfection agents, liposome transfection, electroporation, biolistic particle delivery system (gene gun), microinjection, and the like. In certain embodiments, the reprogramming factor is added to the medium without being coupled to any other component.

[0097] In some embodiments, a first set of reprogramming factors that can be used to reprogram a first type of cell into a second type of cell includes a glycogen synthase kinase 3 (GSK3) inhibitor, a transforming growth factor-β (TGFβ) inhibitor, and a cyclic AMP inducer. In other embodiments, the first set of reprogramming factors further includes basic fibroblast growth factor (bFGF), a DNA methyltransferase inhibitor, a histone methyltransferase inhibitor (e.g., a DOT1L inhibitor), a histone deacetylase inhibitor, BMP4, or a combination thereof.

[0098] In some embodiments, the first set of reprogramming factors consists of a GSK3 inhibitor, a TGFβ inhibitor, and a cyclic AMP inducer. In some embodiments, the first set of reprogramming factors consists of a GSK3 inhibitor, a TGFβ inhibitor, a cyclic AMP inducer, and bFGF. In some embodiments, the first set of reprogramming factors consists of a GSK3 inhibitor, a TGFβ inhibitor, a cyclic AMP inducer, a DNA methyltransferase inhibitor, a histone deacetylase inhibitor, and BMP4. In some embodiments, the first set of reprogramming factors consists of a GSK3 inhibitor, a TGFβ inhibitor, a cyclic AMP inducer, a DNA methyltransferase inhibitor, a histone methyltransferase inhibitor (e.g., a DOT1L inhibitor), and a histone deacetylase inhibitor.

[0099] In some embodiments, reprogramming factors can sometimes be functionally replaced by paralogs within their respective families.

[0100] As used herein, the term "inhibitor" refers to an agent that can reduce the expression and / or activity of a targeted expressed product (e.g., the mRNA encoding the target or the target polypeptide) by, for example, at least 10% or more (e.g., 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more). The efficacy of an inhibitor (e.g., its ability to reduce the target level and / or activity) can be determined, for example, by measuring the level of the expressed product and / or the activity of the target. Methods for measuring the level of a given mRNA and / or polypeptide are known to those of skill in the art. For example, RT-PCR can be used to determine RNA levels, and Western blotting using an antibody can be used to determine polypeptide levels. Target activity can be measured using methods known in the art and described herein, such as transcriptional activity assays. In some embodiments, the inhibitor can be an inhibitory nucleic acid; an aptamer; an antibody or a binding fragment thereof; or a small molecule.

[0101] GSK3 (glycogen synthase kinase 3) is a serine / threonine protein kinase that is involved in many signaling pathways related to glycogen production, apoptosis, stem cell maintenance, and so on. GSK3 includes isoforms (GSK3α and GSK3β) encoded by different genes and having high homology at the amino acid level. Examples of GSK3 inhibitors include GSK3α inhibitors and GSK3β inhibitors. Specific examples of GSK3 inhibitors include siRNA against the gene encoding GSK3, anti-GSK3 antibodies, CHIR98014 (Millipore, Bedford), CHIR99021 (Millipore), Kenpaullone (Millipore), AR-AO144-18 (Santa Cruz Biotechnology, Santa Cruz), TDZD-8 (Abcam, Cambridge, US), SB216763 (Abcam, Cambridge, US), BIO ((2’Z,3’E)-6-bromoindirubin-3’-oxime) (R&D Systems, Minneapolis, US), TWS-119 (Abcam, Cambridge, US), SB415286 (Abcam, Cambridge, US), Ro3303544 (US6479490), LiCl, Li2CO3, and so on. All of these are commercially available or can also be prepared by those of ordinary skill in the art with reference to known literature.

[0102] In certain embodiments, the GSK3 inhibitor is selected from the group consisting of CHIR99021, LiCl, Li2CO3, and BIO. In certain embodiments, the GSK3 inhibitor is CHIR99021. In certain embodiments, the GSK3 inhibitor is BIO. The concentration of the GSK3 inhibitor in the medium is appropriately determined according to the type of inhibitor to be used. In the case of CHIR99021 or BIO, the concentration is generally 0.1 - 10 μM, preferably 1 - 5 μM, more preferably about 3 μM. One or more categories of GSK3 inhibitors can be used in combination.

[0103] TGFβ (transforming growth factor-β) is a multifunctional cytokine belonging to the transforming growth factor superfamily. TGFβ includes three different mammalian isoforms (TGFβ1 to 3, HGNC symbols TGFBETA1, TGFBETA2, TGFBETA3). TGFβ can be secreted by many cell types including macrophages and is in a latent form, in which TGFβ is complexed with two other polypeptides, latent TGFβ binding protein (LTBP) and latent associated peptide (LAP). The source of the TGFβ inhibitor to be used in the present invention is not particularly limited as long as it can effectively inhibit TGFβ function. TGFβ inhibitors are commercially available or can also be prepared by those of ordinary skill in the art with reference to known literature. Specific examples of TGFβ inhibitors include siRNA against the gene encoding GSK3, anti-TGFβ antibodies, and chemical antagonists.

[0104] In certain embodiments, the TGFβ inhibitor is selected from the group consisting of: SB431542 (Tocris Bioscience, Bristol, UK), Repsox (Tocris Bioscience, Bristol, UK), LDN193189 (Tocris Bioscience, Bristol, UK), and tranilast (Rizaben). In certain embodiments, the TGFβ inhibitor is SB431542. The concentration of the TGFβ inhibitor in the medium is appropriately determined according to the type of inhibitor to be used. In the case of SB431542, the concentration is generally 0.1 - 20 μM, preferably 1 - 10 μM, more preferably about 5 μM. In the case of Repsox, the concentration is generally 0.1 - 20 μM, preferably 1 - 15 μM, more preferably about 10 μM.

[0105] As used herein, the term "cyclic AMP inducer" refers to any compound that increases the intracellular concentration of cAMP in cells by at least 2%, preferably at least 5%, more preferably at least 10%, and most preferably at least 20% at an effective concentration. Methods for measuring intracellular cAMP levels are known to those of skill in the art. Preferred cyclic AMP inducers include isobutylmethylxanthine and forskolin. The concentration of forskolin is generally 1 - 20 μM, preferably 5 - 15 μM, more preferably about 10 μM.

[0106] bFGF (basic fibroblast growth factor) is a type of protein that inherently exists in the body and is known to control cell growth and differentiation and has functions such as angiogenesis, smooth muscle cell proliferation, wound healing, tissue repair, hematopoiesis, nerve cell differentiation, etc. in various tissues and organs. The origin of bFGF to be used in the present invention is not particularly limited as long as it is effective for reprogramming. bFGF is commercially available or can also be prepared by those of ordinary skill in the art with reference to known literature. For example, it can be synthesized based on known base sequences and amino acid sequences. For example, the amino acid sequences can be obtained according to NCBI accession numbers AAA52448.1 (human) and AAA37621.1 (mouse). The concentration of bFGF in the medium is generally 1 - 50 ng / ml, preferably about 1 - 20 ng / ml, more preferably about 10 ng / ml or about 20 ng / ml.

[0107] Histone acetylation is a reversible modification, and deacetylation is catalyzed by an enzyme family called histone deacetylases (HDACs). HDAC inhibitors include valproic acid (VPA), trichostatin A (TSA), vorinostat (suberoylanilide hydroxamic acid, SAHA, Merck & Co., Inc.), depsipeptide (romidepsin, FK-228, Gloucester Pharmaceutical Inc.), trapoxin, depudecin, FR901228 (Fujisawa Pharmaceuticals), and butyrate. In certain embodiments, the HDAC inhibitor is VPA. The concentration of the HDAC inhibitor in the medium is appropriately determined according to the type of inhibitor to be used. In the case of VPA, the concentration is generally 1 - 2000 μM, preferably 10 - 1000 μM, more preferably about 500 μM.

[0108] The basal medium supplemented with the first set of reprogramming factors is renewed every 1, 2, or 3 days.

[0109] In certain embodiments, the first type of cell is cultured in the presence of a first set of reprogramming factors for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 days, 1.5 months, or 2 months. In certain embodiments, the first type of cell is cultured in the presence of a first set of reprogramming factors for no more than 2 months, 1.5 months, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day. In certain embodiments, the first type of cell is cultured in the presence of a first set of reprogramming factors for 1 day to 2 months, 1 day to 1 month, 1 day to 25 days, 1 day to 20 days, 1 day to 19 days, 1 day to 18 days, 1 day to 17 days, 1 day to 16 days, 5 days to 16 days, 7 days to 16 days, 8 days to 16 days, 9 days to 16 days, 10 days to 16 days, 11 days to 16 days, 12 days to 16 days, 1 day to 15 days, 1 day to 14 days, 1 day to 13 days, 1 day to 12 days, 2 days to 12 days, 3 days to 12 days, 4 days to 12 days, 5 days to 12 days, 6 days to 12 days, 7 days to 12 days.

[0110] In one aspect, the present disclosure provides a method for reprogramming a second type of cell into a third type of cell, comprising culturing the second type of cell in the presence of a second set of reprogramming factors, wherein the second set of reprogramming factors comprises a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0111] Casein kinase 1 (CK1) is a serine / threonine-selective enzyme that acts as a regulator of signal transduction pathways in most eukaryotic cell types. CK1 isoforms are involved in Wnt signaling, circadian rhythms, nucleocytoplasmic shuttling of transcription factors, DNA repair, and DNA transcription. The origin of the CK1 inhibitor to be used in the present invention is not particularly limited as long as it is effective for reprogramming. CK1 inhibitors are commercially available or can also be prepared by those of ordinary skill in the art with reference to known literature. Specific examples of CK1 inhibitors include siRNA against the gene encoding CK1, anti-CK1 antibodies, and chemical antagonists. Preferably, the CK1 inhibitor is CKI-7. The concentration of the CK1 inhibitor in the medium is appropriately determined according to the type of inhibitor to be used. In the case of CKI-7, the concentration is generally 0.1 - 20 μM, preferably 1 - 10 μM, more preferably about 5 μM. Other inhibitors of casein kinase 1 include PF 670462 (AndiBio, Minneapolis, USA), D4476 (AndiBio, Minneapolis, USA), (R)-CR8 (AndiBio, Minneapolis, USA), (R)-DRF053 dihydrochloride (AndiBio, Minneapolis, USA), TAK 715 (AndiBio, Minneapolis, USA), PF 4800567 hydrochloride (AndiBio, Minneapolis, USA), LH 846, CKI 7 dihydrochloride (AndiBio, Minneapolis, USA), SR3029 (AndiBio, Minneapolis, USA), Epiblastin A (AndiBio, Minneapolis, USA), PF 5006739 (AndiBio, Minneapolis, USA).

[0112] In certain embodiments, the second type of cell is an ectodermal cell. In certain embodiments, the ectodermal cell is a neural crest cell (NC) or an NCC-like cell. In certain embodiments, the second type of cell is an NC or an NCC-like cell. NC or NCC-like cells can be differentiated from human embryonic stem cells (hES cells) using, for example, dual SMAD inhibitors as described herein or as described in WO / 2010 / 096496. NC or NCC-like cells can be differentiated from hES cells using a combination of a Wnt agonist (e.g., Wnt3a and / or (2’Z,3’E)-6-bromoindirubin-3’-oxime (BIO)) and a SMAD inhibitor (such as SB431542 and / or Noggin); see Menendez et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS), November 29, 2011, Vol. 108, No. 48, 19240-19245. For example, efficient induction of NC or NCC-like cells has been reported after contacting hES cells with SB431542 and (2’Z,3’E)-6-bromoindirubin-3’-oxime (BIO) (in the presence or absence of Noggin) or Wnt3a and SB431542. NC can also be obtained from neural rosette cultures, for example, by culturing hES cells on MS5 stromal feeder cells (see Lee et al., Stem Cells 25(8), 1931-1939 (2007), which is incorporated herein by reference in its entirety). NC can also be obtained from a number of tissues, including in the developing embryo, neural tube, sciatic nerve, intestine, and dorsal root ganglia; as well as in the larva and adult, dorsal root ganglia, bone marrow, skin, heart, cornea, tooth, and carotid body. See Nagoshi et al., Journal of Cellular Biochemistry 107:1046-1052 (2009); Crane and Trainor, Annual Review of Cell and Developmental Biology 2006.22:267-86; and Blum, Brain Research Bulletin 83(2010)189-193, each of which is incorporated herein by reference in its entirety. In certain embodiments, NCC-like cells are obtained by reprogramming the first type of cell according to the present application.

[0113] In some embodiments, culturing a second type of cell in the presence of a second set of reprogramming factors causes at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the cells to become a third type of cell.

[0114] In certain embodiments, the third type of cell is an ectodermal cell. In certain embodiments, the third type of cell is a neuronal cell. In certain embodiments, the third type of cell is a corneal endothelial cell-like cell (CEC-like cell).

[0115] "Corneal endothelial cell" or "CEC" generally refers to mitochondrion-rich cells that line the posterior surface of the cornea and face the anterior chamber of the eye in a living organism. To distinguish the chemically induced CECs (ciCECs) provided herein from primary CECs, the resulting CECs are also named corneal endothelial cell-like cells (CEC-like cells). The CEC-like cells obtained by the reprogramming methods disclosed herein can be identified or recognized by their exhibiting one or more of the following endogenous CEC properties: expressing CEC markers, the ability to form a monolayer of uniformly sized cells with a predominantly hexagonal shape, the ability to form a "leaky pump" (allowing solutes and nutrients to leak from the aqueous humor into the more superficial layers of the cornea and simultaneously actively pumping water in the opposite direction from the stroma to the aqueous humor). Exemplary CEC markers include, but are not limited to: Na + / K + ATPase, tight junction protein 1 (TJPl / ZO-1), KLF13, AQP1, collagen VIII, SLC 16A3, CFTR, NBC1, CA2, AE2 / SCL4A2, SCL16A1, CA12, CA4, FoxCl. For example, CECs typically express collagen VIII, the Na+K+ATPase pump, and ZO-1, and do not express vWF and CD31 (the latter being present in vascular endothelial cells). Additionally, CECs may express one or more corneal endothelial pump markers (which include AQP1, CA2, CA4, CA12, SCL14A2, SLC 16A1, SLC 16A3, SLC 16A7, CFTR, NHE1, ADC Y10, voltage-dependent anion channels VDAC2 and VDAC3, chloride channel protein CLCN2 and CLC), perioptic neural crest markers (which include PITX2 and FOXCl) and / or cell adhesion

[0116] Adhesion and matrix proteins (including Occludin, Connexin 43, 9.3E antigen, collagen III, collagen IV, N-cadherin, VE-cadherin, E-cadherin, β-catenin, laminin α4, Nidogen-2, and Netrin 4). For example, CECs can express at least one corneal endothelial pump marker, at least one periocular neural crest marker, and at least one cell adhesion and matrix protein.

[0117] In some embodiments, the resulting CEC-like cells exhibit one or more biomarkers consistent with the primary CEC phenotype. In some embodiments, the resulting CEC-like cells express tight junction protein 1 (TJPl / ZO-1), N-cadherin, and Na + / K + ATPase. In some embodiments, the expression of CEC biomarkers is increased by more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000-fold or more relative to the NCC or NCC-like cells from which the CEC-like cells are generated.

[0118] The markers can be detected or measured by methods known in the art. For example, the adhesion junctions formed by N-cadherin can be confirmed by examining the expression at the protein level (e.g., using methods based on antigen-antibody reactions) or the gene level (e.g., using RT-PCR). The Na+ / K+-ATPase pumping function of cells can be measured, for example, according to the methods described in Investigative Ophthalmology & Visual Science, 2010, Vol. 51, No. 8, 3935-3942, and Current Eye Research, 2009, Vol. 34, 347-354, and using an Ussing chamber.

[0119] In certain embodiments, the second set of reprogramming factors further comprises BMP4 and / or a DNA methyltransferase inhibitor.

[0120] Bone morphogenetic proteins (BMPs) are a group of growth factors that promote bone and cartilage formation. BMPs interact with specific receptors on the cell surface called bone morphogenetic protein receptors (BMPRs). Signal transduction through BMPRs causes mobilization of members of the SMAD family of proteins. The signaling pathway involving BMPs, BMPRs, and Smads is important in heart, central nervous system, and cartilage development, as well as postnatal bone development. It plays an important role in embryonic patterning and early skeletal formation during embryonic development. Thus, interfering with BMP signaling affects the body plan of the developing embryo. Examples of BMP inhibitors include, but are not limited to, DMH2 (MilliporeSigma, USA), Dorsomorphin (Abcam, Cambridge, USA), LDN193189 (Tocris Bioscience, Bristol, UK), DMH-1 (Tocris Bioscience, Bristol, UK), K 02288 (Tocris Bioscience, Bristol, UK), and ML 347 (Tocris Bioscience, Bristol, UK). The concentration of the BMP inhibitor in the medium is appropriately determined according to the type of inhibitor to be used, such as 0.1 nM - 10 μM, 0.1 nM - 5 μM, 0.1 nM - 2.5 μM, 0.1 nM - 2 μM, 0.5 nM - 2 μM, 1 nM - 2 μM, 1 nM - 1.5 μM, 1 nM - 1000 nM, 5 nM - 1000 nM, 10 nM - 1000 nM, 50 nM - 1000 nM, 50 nM - 500 nM, 50 nM - 200 nM, 100 nM - 200 nM, 100 nM - 150 nM, or 100 nM.

[0121] BMP4 (bone morphogenetic protein 4) is a member of the bone morphogenetic protein family that is part of the transforming growth factor-β superfamily. BMP4 is found in the ventral marginal zone and in early embryonic development in the eye, heart blood, and otic vesicle. The origin of the BMP4 inhibitor to be used in the present invention is not particularly limited as long as it is effective for reprogramming. The BMP4 inhibitor is commercially available or can also be prepared by those of ordinary skill in the art with reference to known literature. Specific examples of the BMP4 inhibitor include siRNA against the gene encoding BMP4, chordin, noggin, and anti-BMP4 antibody. The concentration of the BMP4 inhibitor in the medium is appropriately determined according to the type of inhibitor to be used, such as 0.01-100 μg / ml, 0.01-50 μg / ml, 0.01-25 μg / ml, 0.01-10 μg / ml, 0.01-5 μg / ml, 0.01-1 μg / ml, 0.01-0.5 μg / ml, 0.01-0.1 μg / ml, 0.01-0.05 μg / ml, 0.05-10 μg / ml, 0.1-10 μg / ml, 0.1-5 μg / ml, 0.1-4 μg / ml, 0.1-3 μg / ml, or 0.1-2 μg / ml, or 10 ng / ml.

[0122] Enzymes of the DNMT (DNA methyltransferase) family can catalyze the transfer of methyl groups to DNA. DNA methylation provides a wide variety of biological functions. The origin of the DNMT inhibitor to be used in the present invention is not particularly limited as long as it is effective for reprogramming. The DNMT inhibitor is commercially available or can also be prepared by those of ordinary skill in the art with reference to known literature. Specific examples of the DNMT inhibitor include siRNA against the gene encoding DNMT, anti-DNMT antibody, and chemical antagonists.

[0123] In certain embodiments, the DNMT inhibitor is selected from the group consisting of: decitabine (Bristol Myers Squibb, UK), 5-azacytidine (Bristol Myers Squibb, UK), 5-aza-dC (Bristol Myers Squibb, UK), and RG108 (Bristol Myers Squibb, UK). The concentration of the DNMT inhibitor in the medium is appropriately determined according to the type of inhibitor to be used, such as 0.1-100 μM, 0.1-50 μM, 0.1-25 μM, 0.1-10 μM, 0.5-10 μM, 1-10 μM, 5 μM.

[0124] DOT1L (Disruptor of Telomeric Silencing 1-Like Protein) is a class of histone methyltransferases (HMTs) that catalyze the methylation of lysine 37 of histone 3 within chromatin structures such as telomeric chromatin. The DOT1L inhibitors to be used in the present invention are not particularly limited as long as they are effective for reprogramming. DOT1L inhibitors are commercially available or can also be prepared by those of ordinary skill in the art with reference to known literature. Specific examples of DOT1L inhibitors include siRNAs against the gene encoding DOT1L, anti-DOT1L antibodies, and chemical antagonists. In certain embodiments, the DOT1L inhibitor is a small molecule DOT1L inhibitor. Without wishing to be bound by theory, it is known that DOT1L can catalyze H3K27 methylation using S-adenosylmethionine (AdoMet) as a cofactor through the AdoMet binding site on DOT1L. Thus, any chemical that mimics the molecular structure of AdoMet and can displace AdoMet from its binding site on DOT1L is contemplated as a DOT1L inhibitor in the present disclosure. In certain embodiments, the DOT1L inhibitor is selected from the group consisting of EPZ004777, EPZ5676 (also known as pinometostat), SGC 0946, and SYC-522.

[0125] In certain embodiments, the second type of cell is cultured in the presence of the second set of reprogramming factors for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 days, 1.5 months, or 2 months. In certain embodiments, the second type of cell is cultured in the presence of the second set of reprogramming factors for no more than 2 months, 1.5 months, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day. In certain embodiments, the second type of cell is cultured in the presence of the second set of reprogramming factors for from 1 day to 2 months, 1 day to 1 month, 1 day to 25 days, 1 day to 20 days, 1 day to 19 days, 1 day to 18 days, 1 day to 17 days, 1 day to 16 days, 5 days to 16 days, 7 days to 16 days, 8 days to 16 days, 9 days to 16 days, 10 days to 16 days, 11 days to 16 days, 12 days to 16 days, 1 day to 15 days, 1 day to 14 days, 1 day to 13 days, 1 day to 12 days, 2 days to 12 days, 3 days to 12 days, 4 days to 12 days, 5 days to 12 days, 6 days to 12 days, 7 days to 12 days.

[0126] In one aspect, the present disclosure provides a method of reprogramming a first type of cell into a third type of cell, comprising the steps of: (a) culturing the first type of cell in the presence of a first set of reprogramming factors, wherein the first set of reprogramming factors comprises a glycogen synthase kinase 3 (GSK3) inhibitor, a transforming growth factor (TGFβ) inhibitor, and a cyclic AMP inducer; and (b) culturing the cells obtained from step (a) in the presence of a second set of reprogramming factors, wherein the second set of reprogramming factors comprises a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0127] In one aspect, the present disclosure provides a method of reprogramming a first type of cell into a third type of cell, comprising culturing the first type of cell in the presence of a first set of reprogramming factors and a second set of reprogramming factors, wherein the first set of reprogramming factors comprises a glycogen synthase kinase 3 (GSK3) inhibitor, a transforming growth factor (TGFβ) inhibitor, and a cyclic AMP inducer, and the second set of reprogramming factors comprises a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0128] In certain embodiments, the first set of reprogramming factors further comprises bFGF. In certain embodiments, the second set of reprogramming factors further comprises BMP4 and / or a DNA methyltransferase inhibitor.

[0129] In some embodiments, culturing the first type of cell in the presence of the first set and the second set of reprogramming factors results in at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the cells becoming the third type of cell.

[0130] In certain embodiments, the method further comprises washing the cells obtained from step (a) prior to commencing step (b). In certain embodiments, there is no washing step between step (a) and step (b).

[0131] C. Pharmaceutical Compositions and Therapeutic Methods

[0132] In one aspect, the present disclosure provides a population of NCC-like cells produced according to the methods provided herein. In certain embodiments, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the population are NCC-like cells.

[0133] In one aspect, the present disclosure provides a method for generating patient-specific neural crest cell-like cells. In one embodiment, the first type of cell is obtained from a subject suffering from a neurological disorder.

[0134] In one aspect, the present disclosure provides a population of corneal endothelial cell-like cells (CEC-like cells) produced according to the methods provided herein. In certain embodiments, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the cells in the population of the present disclosure are CEC-like cells.

[0135] In some embodiments, NCC-like cells or CEC-like cells produced according to the methods provided herein are purified ex vivo. In some embodiments, the methods for reprogramming produce high-purity NCC-like cells or CEC-like cells and do not require purification. For example, in some embodiments, the reprogramming methods provided herein can yield a cell composition comprising at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more NCC-like cells or CEC-like cells. In some embodiments, the reprogramming methods provided herein produce NCC-like cells or CEC-like cells of low purity or below the desired purity and require purification. In some embodiments, NCC-like cells or CEC-like cells are purified by substantially separating the NCC-like cells or CEC-like cells from other cells in the composition. In the case of CEC-like cell purification, other cells in the composition can include undifferentiated NCC-like cells and / or NCC-like cells that have differentiated into non-desired cell lineages or phenotypes.

[0136] In one aspect, the present disclosure provides a composition comprising NCC-like cells or CEC-like cells produced according to the methods provided herein. The composition can include one or more pharmaceutically acceptable carriers and diluents.

[0137] The term "pharmaceutically acceptable" indicates that the designated carrier, vehicle, diluent, excipient, and / or salt is generally chemically and / or physically compatible with the other components that make up the formulation and is physiologically compatible with its recipient. Pharmaceutically acceptable carriers for the pharmaceutical compositions disclosed herein can include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antibacterial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestering / chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

[0138] The compositions described herein can further include components facilitating transplantation. The compositions described herein can be pyrogen-free or substantially pyrogen-free and pathogen-free, where pathogens include bacterial contaminants, mycoplasma contaminants, and viruses.

[0139] Compositions comprising the NCC-like cells or CEC-like cells described herein can further comprise an immunosuppressant or an immunotolerant agent.

[0140] In another aspect, the present disclosure relates to the therapeutic use of the CEC-like cells provided herein. For example, the CEC-like cells of the present invention can be used as grafts in cell therapy for treating diseases that require corneal endothelium transplantation, such as bullous keratopathy, corneal edema, corneal leukoma, and the like.

[0141] In one aspect, the present disclosure provides a method for treating a disease or condition associated with dysfunctional or damaged corneal endothelial cells, comprising administering to a subject in need thereof an effective amount of the CEC-like cells provided herein or a composition comprising the CEC-like cells.

[0142] In some embodiments, administering corneal endothelial cells induces the eye's healing process. In some embodiments, administering corneal endothelial cells replenishes diseased tissue. In some embodiments, administering corneal endothelial cells has a regenerative effect on damaged or diseased eye tissue.

[0143] The route of administration can include any suitable means, including but not limited to topical application to the eye site, injection into the eye site, transplantation at the eye site, and the like. In some embodiments, the specific mode of administration selected will depend on the particular treatment, the patient's disease state or condition, the nature of other drugs or therapeutic agents administered to the subject, or the route of administration, among other factors. In some embodiments, the corneal endothelial cells can be administered to the subject in a single dose or in multiple doses at selected time intervals, such as in titrated doses. When administering multiple doses, the doses can be spaced apart, for example, by one week, one month, one year, or ten years. One or more growth factors, hormones, interleukins, cytokines, small molecules, or other cells can also be administered before, during, or after administering the cells to further bias the cells towards a particular cell type.

[0144] As used herein, the term "effective amount" generally refers to the amount of a compound or cell that is sufficient to achieve such treatment for a disease when administered to a patient for treating the disease. The effective amount can be a prophylactically effective amount and / or a preventive effective amount. The effective amount can be an amount that is effective in reducing signs / symptoms, preventing the occurrence of signs / symptoms, reducing the severity of the occurrence of signs / symptoms, eliminating the occurrence of signs / symptoms, slowing the development of the occurrence of signs / symptoms, preventing the development of the occurrence of signs / symptoms, and / or achieving prophylaxis against the occurrence of signs / symptoms. The "effective amount" can vary depending on the disease and its severity, as well as the age, weight, medical history, susceptibility, and pre-existing conditions of the patient to be treated. For the purposes of the present invention, the term "effective amount" is synonymous with "therapeutically effective amount".

[0145] As used herein, "treating / treatment" encompasses the treatment of a disease or medical condition described herein in a subject, such as a human, animal, or mammal, and includes: (i) inhibiting the disease or disorder, i.e., arresting its development; (ii) alleviating the disease or disorder, i.e., causing regression of the disorder; (iii) slowing the progression of the disorder; and / or (iv) inhibiting, alleviating one or more symptoms of the disease or medical condition or slowing the progression of one or more symptoms of the disease or medical condition.

[0146] As used herein, the term "subject" is not limited to a particular species or sample type. By way of example, the term "subject" can refer to a patient and often refers to a human patient. However, the term is not limited to humans and thus encompasses a variety of mammalian species, such as non-human veterinary mammals, such as dogs, cats, rabbits, pigs, rodents, horses, or monkeys.

[0147] The CEC-like cells provided herein are cell aggregates, such as clumps obtained by concentration and filtration and analogs thereof, and the analogs are used as medicaments of the present invention. In addition, it is also possible to add cryoprotectants, such as glycerol, DMSO (dimethyl sulfoxide), propylene glycol, acetamide, etc., to the medicaments and cryopreserve the mixture. In order to use the medicaments more safely, the medicaments can be subjected to treatments under conditions that cause pathological protein denaturation, such as heat treatment, radiation treatment, etc., while retaining the function of corneal endothelial cells.

[0148] In some embodiments, the CEC-like cells can be administered in combination with surgery. In some embodiments, the surgery can be Descemet's stripping with endothelial keratoplasty (DSEK), which includes removing Descemet's membrane and corneal endothelium and subsequently transplanting donor tissue. Alternatively, the surgery can be penetrating keratoplasty (PKP), in which the entire cornea is removed and replaced. Other surgeries can include lamellar keratoplasty, Descemet's Membrane Endothelial Keratoplasty (DMEK), DSAEK, and DLEK.

[0149] "Diseases or conditions associated with dysfunctional or damaged corneal endothelial cells" include any disease or condition suitable for treatment by administration of CEC-like cells, including diseases in which the number of CECs in a subject is reduced or dead, the density is decreased, or otherwise becomes dysfunctional. Primary diseases affecting the corneal endothelium include Fuchs' dystrophy, iridocorneal endothelial syndrome, posterior polymorphous dystrophy, and congenital hereditary endothelial dystrophy. Secondary diseases or conditions for which effective treatment may include replacement of the corneal endothelium include age-related macular degeneration (AMD), retinitis pigmentosa, glaucoma, corneal dystrophy, contact lens use, cataract surgery, and late endothelial failure in corneal transplantation. Corneal endothelial cell diseases additionally include any damage to the cornea, such as damage caused by chemical irritation, damage resulting from contact lens use, reaction or sensitivity (e.g., to contact lens care solutions, cosmetics, eye drops, pharmaceuticals, fumes, etc.), scratching, abrasion, bruising, contusion, foreign bodies in the eye (e.g., sand or dust), or exposure to ultraviolet light (from, for example, sunlight, fluorescent lighting, snow reflection, water reflection, or arc welding or other exposure). In certain embodiments, the disease or condition associated with dysfunctional or damaged corneal endothelial cells results in loss of vision in the subject. In certain embodiments, the loss of vision in the subject is permanent or irreversible.

[0150] In certain embodiments, the corneal endothelial cells are immunocompatible with the subject (e.g., allogeneic or autologous).

[0151] In certain embodiments, the method of treatment may further comprise administering to the subject an immunosuppressive agent or an immunotolerant agent. The immunosuppressive agent or immunotolerant agent may be administered in an amount sufficient to reduce the risk of rejection of the CEC-like cells.

[0152] The immunosuppressive agent or immunotolerant agent may comprise one or more of the following: antilymphocyte globulin (ALG) polyclonal antibody, antithymocyte globulin (ATG) polyclonal antibody, azathioprine, (anti-lL-2Ra receptor antibody), cyclosporin (cyclosporin A), (anti-IL-2Ra receptor antibody), everolimus, mycophenolic acid, (anti-CD20 antibody), sirolimus, tacrolimus, mycophenolate mofetil, and corticosteroids.

[0153] The immunosuppressant can be administered at least about 1, 2, 4, 5, 6, 7, 8, 9, or 10 mg / kg. When an immunosuppressant is used, it can be administered systemically or locally, and it can be administered before, concomitantly with, or after the administration of the CEC-like cells. The immunosuppressive therapy can continue for weeks, months, years, or indefinitely after the cell administration. For example, a patient can receive 5 mg / kg of cyclosporine for 6 weeks after the administration of the CEC-like cells.

[0154] In one aspect, the present disclosure provides the use of the CEC-like cells or compositions provided herein for manufacturing a medicament for treating a disease or condition associated with dysfunctional or damaged corneal endothelial cells.

[0155] In one aspect, the present disclosure provides a method of treating a disease or condition associated with dysfunctional or damaged corneal endothelial cells, comprising administering to a subject in need thereof an effective amount of the NCC-like cells or a composition comprising NCC-like cells provided herein.

[0156] In one aspect, the present disclosure provides the use of the NCC-like cells or compositions provided herein for manufacturing a medicament for treating a disease or condition associated with dysfunctional or damaged corneal endothelial cells.

[0157] The NCC-like cells provided herein can be used to treat neurological diseases.

[0158] As used herein, "neurological disease" is defined as a disorder of the nervous system and includes disorders involving the central nervous system (brain, brainstem, and cerebellum), the peripheral nervous system (including the cranial nerves), and the autonomic nervous system (parts of which are located in the central and peripheral nervous systems). In particular, neurological diseases include any disease in which the function of neural crest cells or Schwann cells is impaired, altered, or disrupted. Examples of neurological diseases associated with Schwann cells are demyelinating diseases, multiple sclerosis, myelopathy, experimental allergic encephalomyelitis (EAE), acute disseminated encephalomyelitis (ADEM), post-infectious or post-vaccination encephalomyelitis, peripheral neuropathy, schwannomatosis, Charcot-Marie- Figure ThreeCharcot-Marie-Tooth disease, Guillain-Barre Syndrome, Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP).

[0159] The present disclosure also provides methods for drug discovery and / or drug screening. Assays for drug discovery and / or drug screening are also provided. In certain embodiments, these methods include administering a drug candidate to NCC-like cells or CEC-like cells and detecting the response of the cells to the drug candidate. Detecting the response can identify whether the drug candidate has suitable properties (e.g., toxicity or therapeutic efficacy). In some embodiments, the methods can be used to determine cell health and viability in the presence of a drug candidate. In some embodiments, the methods can be used to test the toxicity of a drug candidate. In some embodiments, the methods can be used to assess changes in the phenotype of a cell population in the presence of a drug candidate.

[0160] In one aspect, the present disclosure provides a method of using NCC-like cells to screen for a drug that reverses, inhibits, or prevents a neurological disease or a neurological side effect of an agent (e.g., a diabetic agent).

[0161] D. Kits

[0162] In one aspect, the present disclosure provides a kit for reprogramming a first type of cell into a second type of cell, wherein the kit comprises a first set of reprogramming factors, and the first set of reprogramming factors comprises a glycogen synthase kinase 3 (GSK3) inhibitor, a transforming growth factor (TGFβ) inhibitor, and a cyclic AMP inducer.

[0163] In one aspect, the present disclosure provides a kit for reprogramming a second type of cell into a third type of cell

[0164] wherein the kit comprises a second set of reprogramming factors, and the second set of reprogramming factors comprises a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0165] In one aspect, the present disclosure provides a kit for reprogramming a first type of cell into a third type of cell, wherein the kit comprises the first set of reprogramming factors and the second set of reprogramming factors, wherein the first set of reprogramming factors comprises a glycogen synthase kinase 3 (GSK3) inhibitor, a TGFβ inhibitor, and a cyclic AMP inducer, and the second set of reprogramming factors comprises a TGFβ inhibitor and a casein kinase 1 inhibitor.

[0166] The kit may further include an instruction manual and a package that separates each of the components in the kit.

[0167] All publications and patents cited in this specification are incorporated herein by reference in their entirety.

[0168] Examples

[0169] Example 1: Materials and Methods

[0170] 1. Animals

[0171] All animal experiments were approved by the Animal Ethics Committee of Wenzhou Medical University, Wenzhou, China. Oct4-GFP transgenic allele-bearing mice (CBA / CaJ×C57BL / 6J) were obtained from the Jackson Laboratory; Wnt1 - cre and Fsp1 - Cre mice were from the Jackson Laboratory (BALB / c - Tg(S100a4 - cre)1Egn / YunkJ); ROSA26 - tdTomato mice were from the Jackson Laboratory (Gt(ROSA)26Sortm14(CAG - tdTomato)Hze). 129Sv / Jae and C57BL / 6 mice were from Beijing Vital River Laboratory. Wnt1 - Cre / ROSA26 tdTomato and Fsp1 - Cre / ROSA26 tdTomato mice were obtained by crossing Wnt1 - Cre and Fsp1 - Cre mice with ROSA26 - tdTomato mice (see Figure 4A). New Zealand white rabbits were obtained from JOINN Laboratories (Suzhou) Inc., Suzhou, China. All animals were handled in accordance with the Statement for the Use of Animals in Ophthalmic and Vision Research of the Association for Research in Vision and Ophthalmology (ARVO). All animals were housed under stable conditions (21°C ± 2°C) with a 12-hour dark / light cycle.

[0172] 2. Cell culture

[0173] As previously described, primary mouse embryonic fibroblasts (MEFs) were isolated from mouse embryos at embryonic day 13.5 (E13.5) (Liu, C., Hu, X., Li, Y., Lu, W., Li, W., Cao, N., Zhu, S., Cheng, J., Ding, S. and Zhang, M. (2019). "Conversion of mouse fibroblasts into oligodendrocyte progenitor-like cells through a chemical approach". Journal of molecular cell biology; Wang, H., Cao, N., Spencer, C.I., Nie, B., Ma, T., Xu, T., Zhang, Y., Wang, X., Srivastava, D. and Ding, S. (2014). "Small molecules enable cardiac reprogramming of mouse fibroblasts with a single factor, Oct4". Cell reports 6, 951-960). Briefly, the head, tail, limbs, and visceral tissues of the embryo were carefully removed and discarded. The remaining tissues were minced into small pieces, trypsinized with 0.25% trypsin / EDTA (Gibco) and plated onto 10-cm culture dishes. The fibroblasts were cultured in fibroblast medium containing DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mM GlutaMAX (Gibco), 0.1 mM non-essential amino acids (Sigma), 100 units / mL penicillin, and 100 mg / mL streptomycin (Gibco). All fibroblasts were expanded for two passages and then used for further experiments. To prepare Wnt1-MEFs, fibroblasts derived from Wnt1 - Cre / ROSA26 tdTomato mouse embryos were sorted for tdTomato - cells by FACS.

[0174] Mouse primary corneal endothelial cells (CECs) (CP-M179) and culture medium (CM-M179) were purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China). Mouse primary CECs were cultured in DMEM containing 10% FBS (Gibco), 0.1 mM non-essential amino acids (Sigma), 2 mM GlutaMAX (Gibco), and 1% penicillin-streptomycin (Gibco). mESCs were maintained in ESC medium, which consisted of DMEM containing 10% FBS (Gibco), LIF, 0.1 mM non-essential amino acids (Sigma), 2 mM GlutaMAX (Gibco), 1% penicillin-streptomycin (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), CHIR99021 (3 mM), and PD0325901 (1 mM).

[0175] Human embryonic fibroblasts (HEFs), human neonatal fibroblasts (HNFs), and human adult fibroblasts (HAFs) were purchased from ScienCell Research Laboratories. Human umbilical cord mesenchymal stem cells (MSCs) were kindly provided by Nuwacell Ltd. (Hefei, China). The cells were maintained in fibroblast medium containing DMEM (Gibco) supplemented with 10% FBS (Gibco), 1% GlutaMAX (Gibco), and 1% NEAA (Gibco). Human urinary cast epithelial cells (UCs) were derived from 100 - 300 mL urine samples from normal individuals as previously reported (Zhou, T. et al. (2012). Generation of human induced pluripotent stem cells from urine samples. Nature Protocols 7, 2080 - 2089).

[0176] 3. FACS sorting for generating tdMEFs

[0177] For the preparation of tdMEFs, the resulting fibroblasts were sorted for tdTomato + / p75 - cells by FACS sorting. Primary MEFs were obtained from Fsp1 - Cre / Rosa26 tdTomato (Fsp1 - Cre mice × Rosa26 tdTomatoIsolated from E13.5 mouse embryos with the genetic background of (mouse). Passage 2 MEFs were dissociated with 0.25% trypsin at 37 °C for 5 minutes and neutralized with MEF medium. Those MEFs were stained with a specific antibody against p75 and underwent FACS sorting for tdTomato + / p75 - cells. During FACS sorting, Matrigel-coated 24-well culture plates were pre-warmed at 37 °C for at least 30 minutes, followed by inoculation of tdMEFs. Immediately after FACS sorting, tdMEFs were seeded at 15,000 cells / well into pre-warmed Matrigel-coated 24-well culture plates in MEF medium supplemented with 1 μM Thiazovivin (Tzv) at 37 °C in 5% CO2 and 20% O2 for 5 hours to allow MEFs to attach to the plate. After 5 hours, the medium was changed to MEF medium without Tzv, and the tdMEFs were cultured overnight at 37 °C in 5% CO2.

[0178] 4. Small molecule compounds and libraries

[0179] Small molecules were obtained from Sigma, including the GSK3b inhibitor CHIR 99021 (SML1046), the TGFb inhibitor SB431542 (S4317), the DNA methylation inhibitor 5-aza-dC (A3656), the cyclic AMP inducer forskolin (F6886), and CKI-7 (C0742). bFGF was obtained from Peprotech. The DOT1L inhibitor EPZ004777 (S7353) and the ROCK inhibitor Y-27632 (S1049) were obtained from Selleck.

[0180] 5. Immunocytochemistry

[0181] To further study the expression of typical neural crest (NC) cell markers, reprogrammed MEFs were fixed, immunostained, and analyzed. Briefly, the cells were washed once with 1×PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature, followed by permeabilization with 1×PBS solution containing 0.2% Triton X-100 for 10 minutes and blocking with 7.5% BSA for at least 1 hour. All primary antibodies were diluted in 7.5% BSA and incubated overnight at 4°C. The cells were washed with 1×PBS for 10 minutes at room temperature, five times. Secondary antibodies Alexa-488, Alexa-555, and Alexa-647 were purchased from Invitrogen, diluted in 7.5% BSA, and incubated for 1 hour at room temperature, followed by five 10-minute washes with 1×PBS. The nuclei were stained with DAPI. The antibodies used in this study are listed in Table 2.

[0182] Table 1: Antibodies used in this study

[0183]

[0184]

[0185] 6. Statistical analysis

[0186] All experiments were performed independently at least three times. Results are expressed as mean ± SD. Data were analyzed by unpaired two-tailed Student’s t-test to compare two groups and by one-way ANOVA and Tukey’s test or Dunnett’s multiple comparisons test to compare multiple groups. All analyses were performed using SPSS Statistics 19.0 software. A P value < 0.05 was considered significant.

[0187] 7. Cell cycle analysis

[0188] The cells were carefully dissociated into single-cell suspensions using Accutase solution, washed twice with PBS, and then fixed overnight with cold 70% ethanol. The fixed cells were washed twice with PBS, followed by ribonuclease (100 μg / mL, Sigma) treatment and propidium iodide (50 μg / mL, Sigma) staining at 37°C for 30 minutes. Approximately 1×106 Cells were counted to determine the cell cycle distribution pattern. The percentages of cells in the G1, S, and G2 / M phases of the cell cycle were analyzed using ModFit 4.1 (Verity Software House).

[0189] 8. Transplantation

[0190] Rabbits weighing 2.0 - 2.5 kg were anesthetized intramuscularly with ketamine hydrochloride (60 mg / kg) and xylazine (10 mg / kg, Bayer, Munich, Germany). The rabbits were divided into 2 groups (n = 10 per group), and their right eyes were used for this experiment. After disinfecting and aseptically covering the operative site, a 6-mm corneal incision centered at 12 o'clock was made with a diamond knife, and viscoelastic agent (Healon; Amersham Pharmacia Biotech AB) was infused into the anterior chamber. After marking the corneal surface with a marker pen (Devon Industries, Madrid, Spain), a 6.0-mm diameter circular opening for descemetorhexis was created at the corneal center with a 30-gauge needle (Terumo, Tokyo, Japan), and Descemet's membrane was removed from the anterior chamber of the eye. As previously described, the corneal endothelium was mechanically scraped from Descemet's membrane using a lacrimal duct irrigator (Shandong Weigao). Fsp ciCEC was dissociated using 0.25% trypsin - EDTA, resuspended in basal medium at a density of 1 × 10 - cells / ml, and kept on ice. The anterior chamber was washed three times with PBS. After this procedure, a 26-gauge needle was used to inject 1 × 10 7 cells suspended in 100 μl of basal DMEM containing 100 μM ROCK inhibitor Y-27632 (Selleckchem). 6Individual cultured ciCECs were injected into the anterior chamber of the right eye. Subsequently, rabbits in the cryo-alone (isolated cryoinjury), CE eye-down (cryoinjury and CE injection), and globe eye-down (cryoinjury and globe injection) groups were kept in the eye-down position for 24 hours so that the cells could attach by gravity, and rabbits in the globe eye-up group were kept in the eye-up position under deep anesthesia for 24 hours. Each operated eye was examined externally, twice or three times a week, and photographs were taken on days 3, 7, 14, and 28 after injection. At 0.5, 1, 3, 7, 14, 21, and 28 days after surgery, the central corneal thickness was measured using an ultrasound pachymeter, and the intraocular pressure was measured using a pneumatic tonometer. The average of three readings was taken.

[0191] 9. Cell proliferation assay

[0192] Via Click-iT TM Ethynyl deoxyuridine (EdU) Alexa Fluor 488 imaging kit (Invitrogen), the proliferation rate of ciCECs cultured in either differentiation medium alone or M5 alone was determined according to the manufacturer's instructions. Briefly, passage CECs were seeded onto slides at a low density of 5×10 3 cells / cm² and cultured for 24 hours.

[0193] 10. Transmission electron microscopy (TEM) analysis

[0194] For TEM analysis, cells were fixed in 2.5% EM-grade glutaraldehyde (Servicebio) at 4 °C for 2 - 4 hours, washed with 0.1 M phosphate buffer (pH 7.4), post-fixed in 1% osmium tetroxide at 4 °C for 2 - 4 hours, washed and then dehydrated in a series of ethanol (50 - 100%) to a final rinse in 100% acetone, followed by incubation in 1:1 acetone / Pon 812 (SPI) for 2 hours and in 1:2 acetone / Pon 812 overnight. Samples were embedded in Pon 812, polymerized at 60 °C for 48 hours, and then sectioned (60 - 80 nm) with a diamond knife (Daitome). Sections were stained with 2% uranyl acetate, followed by lead citrate staining, and visualized using an HT7700 transmission electron microscope (HITACHI).

[0195] 11. Karyotype analysis

[0196] Cells were treated with 0.1 μg / mL colchicine (Gibico) at 37 °C for 2 hours, trypsinized, resuspended and incubated at 37 °C in 0.075 M potassium chloride for 15 minutes, fixed with 3:1 methanol:acetic acid, and then dropped onto slides to disperse the chromosomes. Chromosomes were visualized by staining with Giemsa (Saiweier Biology).

[0197] 12. RNA Sequencing and Analysis

[0198] Total RNA from each sample was isolated using TRIzol reagent and purified using the RNeasy 23 Micro Kit (Qiagen) according to the manufacturer's instructions. RNA quality and quantity were assessed using NanoDrop 2000, Agilent 2100 Bioanalyzer, and Agilent RNA6000 Nano Kit. RNA library construction and RNA sequencing were performed by Annoroad Gene Technology. Sequencing libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina24 (NEB) following the manufacturer's recommendations, and library clustering was performed using the HiSeq PE Cluster Kit v4-cBot-HS (Enlighten Company). After cluster generation, the libraries were sequenced on the Enlighten Company platform and 150 bp paired-end reads were generated. Initial data analysis was performed on BMKCloud (http: / / www.biocloud.net / ).

[0199] 13. FACS Cell Measurement

[0200] For MEF preparation, fibroblasts with the desired genotype were cultured in MEF medium until they reached a confluence of over 80%. Cells were washed twice with 1×PBS and treated with 0.25% trypsin at 37 °C for 5 minutes. After harvesting, cells were passed through a 70-μm filter, washed twice with pre-cooled buffer (1×PBS, 1.5% FBS, 0.5% BSA), and resuspended in the buffer. At the recommended concentration, cells were incubated with FITC-conjugated P75 antibody (Abcam) or isotype control (BD) on ice for 30 minutes, or at room temperature for 45 minutes, followed by washing six times with FACS buffer. Cells were then resuspended in FACS buffer and sorted using BD FACSAria II.

[0201] Example 2: Generation of ciNCC and ciCEC from Fibroblasts

[0202] 1. Medium Preparation

[0203] Stage I Medium Preparation

[0204] A basal medium containing DMEM / F12 / Glutamax (Gibco), 10% KSR (Gibco), 10% FBS (Gibco), 1% NEAA (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), which is supplemented with small molecule Repsox (10 μM), Chir99021 (10 μM), forskolin (10 μM), and bFGF (10 ng / ml).

[0205] Before adding the first set of reprogramming factor molecules, the basal medium may also contain DMEM / F12 / Glutamax (Gibco), 0.075% bovine serum albumin (BSA) (Gibco), 1% NEAA (Gibco), and 0.1 mM 2-mercaptoethanol (Gibco), and produce comparable or better effects in cell reprogramming (data not shown).

[0206] In addition, the combination of small molecule Repsox (10 μM), Chir99021 (10 μM), and forskolin (10 μM) can achieve a similar induction effect (data not shown). Shake the medium for 30 minutes to ensure complete dissolution.

[0207] Stage II medium preparation

[0208] DMEM / F12 / Glutamax (Gibco), 10% KSR (Gibco), 1% NEAA (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), supplemented with 5 μM SB431542 and 5 μM CKI-7.

[0209] 2. Chemically induce ciCEC from fibroblasts

[0210] To investigate whether small molecules can chemically reprogram fibroblasts into corneal endothelial cells, we designed a two-step method to directly reprogram mouse fibroblasts into neural crest cells and differentiate them into corneal endothelial cells ( Figure 1 A). In Wnt1 - Cre - Rosa Tomato In mice, tdTomato is faithfully expressed in the neural crest (NC) under the control of the Wnt1 gene. Therefore, MEFs were isolated from Wnt1 - Cre / ROSA26 tdTomato mice at embryonic day E13.5, and the neural crest population was labeled with tdTomato expression. We performed fluorescence-activated cell sorting (FACS) to collect tdTomato -Population to exclude any neural crest or progenitor cells. These MEFs were negative for typical NC markers including Sox10, P75, Hnk1, and AP2α (data not shown). Additionally, these MEFs were also negative for typical neural stem cell (NSC) markers including Sox2, Pax6, and Olig2 (data not shown). Methods for inducing ciNCCs from fibroblasts by small molecules have not been reported. To investigate whether small molecules could chemically reprogram fibroblasts into ciNCCs, we selected over 20 small molecules as our potential candidate molecules for neural crest lineage reprogramming based on targeted epigenetic modifications and regulation of neural crest developmental signaling. The candidate small molecules focused on reprogramming factors in three main classes containing three small molecules: (1) TGF-β signaling inhibitors such as Repsox (R), which inhibits mesoderm and endoderm specification; (2) GSK3 inhibitors such as CHIR99021 (C), which promotes neural development, and (3) cyclic AMP inducers such as forskolin (F). These three compounds were combined with basic fibroblast growth factor (bFGF) as a chemically defined medium (hereinafter referred to as RCF) to reprogram mouse embryonic fibroblasts (MEFs) into ciNCCs.

[0211] Briefly, MEFs were seeded at 50,000 cells / well in a 6-well culture plate. After overnight culture, the MEFs were treated in the chemically defined reprogramming medium of RCF. In the RCF medium, many small, dense cell clusters with clear edges rapidly appeared within twelve days ( Figure 1 B). To identify the combination of RCF sufficient to reprogram mouse fibroblasts into chemically induced neural crest cells (ciNCCs), Wnt1 － MEFs were used to observe tdTomato expression. The results showed that the RCF compounds could reprogram MEFs into tdTomato-positive neural crest cell-like cells ( Figure 1 D). To further investigate the expression of typical NC markers, the RCF-treated MEFs were fixed, immunostained, and analyzed. These cells expressed HNK1, P75, and AP2α ( Figure 1 F). Those fibroblast-derived, highly proliferative, and self-renewable Sox10-positive cells were subsequently termed ciNCCs.

[0212] During the reprogramming process, many small, dense cell clusters with clear edges rapidly appeared within 7 days ( Figure 1 B and Figure 1 C). After treatment with Repsox, Chir99021, forskolin, and bFGF, we observed tdTomato + cells after 10 days of treatment (Figure 1 D and Figure 1 E). On day 12, the markers of neural crest cells (NC) (HNK1, P75, and AP2α) were observed (see Figure 1 F). However, in the absence of bFGF, a combination of the small molecules Repsox (10 μM), Chir99021 (10 μM), and forskolin (10 μM) could achieve a similar induction effect (data not shown).

[0213] After 14 days of induction of ciNCC with SB431542 and CKI-7 differentiation medium, ciNCC differentiated into corneal endothelial cell-like cells (ciCEC) (see Figure 3 C). Figure 3 A shows the markers of stage II-induced ciCEC detected by immunofluorescence staining at day 14 (Na + -K + ATPase, AQP1, vimentin, N-cadherin, laminin, and AQP1). Tight junctions of corneal endothelial cells were also observed under transmission electron microscopy.

[0214] To avoid possible contamination of neural crest cells (ciNCC) in the starting MEFs, we performed lineage tracing experiments to trace the origin of ciNCC and ciCEC ( Figure 4 B). In Fsp1 - Cre - RosaTomato mice, tdTomato was faithfully expressed in ciNCC and ciCEC under the control of the Fsp1 gene. The expression of ciNCC markers P75, Hnk1, AP2α, and SOX10 was observed by immunocytochemistry in Fsp1 - ciNCC (see Figure 4 C). Figure 4 D shows the representative morphological changes of Fsp1 - Cre:R26RtdTomato MEFs and ciCEC induced from these MEFs.

[0215] Using the same procedure as described above, human cells (human embryonic foreskin fibroblasts (HEF), human neonatal fibroblasts (HNF), adult fibroblasts (HAF), human umbilical cord mesenchymal stromal cells (MSC), and urine cells (UC)) were induced by chemical factors. Preferably, the chemical factors added in stage I were Repsox (10 μM), Chir99021 (10 μM), forskolin (10 μM), 5-azacytidine (5 μM), VPA (500 μM), and BMP4 (10 ng / ml). The chemical factors added in stage II were 5 μM SB431542 and 5 μM CKI-7, which were consistent with those mentioned above. The induced ciNCC and ciCEC derived from the above cells are shown in Figure 5 in.

[0216] Example 3: Characterization of ciNCC and ciCEC obtained in Example 2

[0217] 1.1 RNA Preparation and RT-PCR

[0218] To confirm the expression of NC genes (including Sox10, P75, Pax3, and Msx1), total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). Briefly, 1 μg of total RNA was used for reverse transcription reaction with the iScript cDNA Synthesis Kit (Bio-Rad), and the resulting cDNA was diluted five times in H2O for PCR use. For semi-quantitative PCR, 1 μl of 1 / 5 diluted cDNA was used as the template for the PCR program: 95°C for 5 minutes, and 35 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, followed by 72°C for 10 minutes. Quantitative PCR was performed following the protocol of the FAST SYBR Green Master Mix (ABI). All PCRs were repeated three times, and the expression of individual genes was normalized relative to the expression of Gapdh. The primer sequences are listed in Table 2.

[0219] Table 2: Primers for qRT-PCR

[0220]

[0221] 1.2 RNA Sequencing and Analysis Pipeline

[0222] To validate the transcriptome analysis of ciNCC, RNA sequencing was performed. The RNA sequencing library was prepared using the Ovation RNA-Seq System v2 kit (NuGEN). Total RNA (50 ng) was reverse transcribed to synthesize the first-strand cDNA using a combination of random hexamers and poly-T chimeric primers. Subsequently, the RNA template was partially degraded by heating, and the second-strand cDNA was synthesized using DNA polymerase. Subsequently, the double-stranded DNA was amplified using single primer isothermal amplification (SPIA). SPIA is a linear cDNA amplification process in which ribonuclease H degrades the RNA in the DNA / RNA heteroduplex at the 5′ end of the double-stranded DNA, after which the SPIA primer binds to the cDNA and the polymerase starts replication by displacing the existing forward strand at the 3′ end of the primer. Subsequently, random hexamers were used to linearly amplify the second-strand cDNA. Finally, the library from the SPIA-amplified cDNA was prepared using the Ultralow V2 Library kit (NuGEN). The RNA sequencing library was analyzed by Bioanalyzer and quantified by QPCR (KAPA). On the HiSeq 2500 instrument (Enlighten Company), three RNA sequencing libraries were pooled on each lane of paired-end 100 bp sequencing. The known adapters and low-quality regions of the reads were trimmed using Fastq-mcf. The sample QC was assessed using FastQC (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ). The reads were aligned to the mouse reference assembly mm9 using Tophat 2.0.13 (Kim, D. et al. 2011. "TopHat 2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions." Genome Biology 14.). Gene-level expression was combined using Subread featureCounts (Liao et al. 2014. "FeatureCounts: an efficient general-purpose program for assigning sequence reads to genomic features." Bioinformatics 30, 923-930.), using the Ensembl gene annotation of mm9.Before processing differential expression, we used RUVSeq to adjust for batch effects (David Risso et al., 2014, “Normalization of RNA-seq data using factor analysis of control genes or samples”. *Nature Biotechnology* 32, 896 - 902). Genes that did not have at least two samples with CPM (counts per million) values between.5 and 5000 were filtered out. Differential expression P-values were calculated using edgeR (Robinson, M.D. et al., 2010, “edgeR: a Bioconductor package for differential expression analysis of digital gene expression data”. *Bioinformatics* 26, 139 - 140). The built-in R function “p.adjust” was used to calculate the FDR with the Benjamini-Hochberg method (Benjamini and Hochberg, (1995), “Controlling the false discovery rate: a practical and powerful approach to multiple testing”. *Journal of the Royal Statistical Society Series B* 57, 289 - 300). Gene ontology analysis was completed via DAVID Bioinformatics Resources 6.7 or ToppGene. Heatmaps were generated via Cluster 3.0 and viewed via Java Treeview. Analysis of the transcriptome revealed that these cells were very similar to mouse NC but different from MEF.

[0223] 1.3 Western blot

[0224] To confirm the expression of NC-specific and CEC-specific markers, Western blotting was performed. Briefly, cells were collected in the presence of phosphatase inhibitors (Cell Signaling Technology) and mixed with an equal volume of 2× SDS-PAGE sample buffer containing DTT. The samples were boiled and clarified by centrifugation. After electrophoresis, the proteins were transferred onto a PVDF membrane. The transfer efficiency was determined by briefly staining the membrane with Ponceau stain. The membrane was blocked with 5% BSA at room temperature for at least 1 hour and incubated overnight at 4 °C with the desired antibody diluted in TBST solution containing 5% BSA. The membrane was washed five times (10 minutes each) with TBST, followed by incubation with an HRP-conjugated secondary antibody diluted in TBSA at room temperature for 1 hour. After washing five times with TBST, the blot was developed using an ECL Plus detection kit. For the reprogramming of fibroblasts into ciNCCs and ciCECs, these results together demonstrated that the chemical mixture had a robust and general effect (data not shown).

[0225] 2. Maintenance and differentiation potential of ciNCCs

[0226] For the maintenance of ciNCCs after purification by FACS sorting, the cells were cultured on poly-D-lysine / laminin-coated culture plates in neural crest medium, which consisted of Neurobasal medium (Gibco) supplemented with 1× N2, 1× B27, 10 μg / ml bFGF, 10 μg / ml EGF, and 10 ng / ml BMP4.

[0227] To characterize the differentiation potential of expanded ciNCCs, they were first cultured under differentiation conditions. Neuronal differentiation was induced by withdrawing FGF2 / EGF and exposing to BDNF, GDNF, NGF, and dibutyryl cyclic AMP (dbcAMP), generating peripheral neurons. By immunostaining, ciNCCs at passage 5 gave rise to Tuj1+ and peripherin+ neurons, and this differentiation potential was well maintained during long-term culture ( Figure 2 A). Schwann cell differentiation, as assessed by S100b and GFAP+ expression, was induced in the presence of CNTF, neuregulin 1b, and dbcAMP.

[0228] For melanocyte differentiation, after 7 days of induction treatment, ciNCCs were cultured in melanocyte differentiation medium (EBM2 basal medium, 5% (v / v) FBS, 100 ng / ml SCF (Life Technologies), 200 nM endothelin 3 (EDN3, Sigma), 50 ng / ml WNT1, 10 ng / ml FGF2, 5 μg / ml insulin, 1 pM cholera toxin, 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA, Sigma), and 10 μM SB431542 (Sigma)). After 3 - 4 weeks of treatment, melanocytes were observed ( Figure 2 A).

[0229] To examine the differentiation potential towards the mesenchymal lineage, we cultured ciNCCs under MSC culture conditions. Under these conditions, cells with mesenchymal morphology and marker expression (CD105+) emerged (data not shown). After an additional month of culture, most cells expressed CD105 and a set of surface markers characteristic of mesenchymal stem cells. We used an established mesenchymal stem cell differentiation protocol and demonstrated that mesenchymal progenitor cells generated from ciNCCs were capable of adipocyte, chondrogenic, and osteogenic ( Figure 2 B) differentiation.

[0230] 3. Functional Characterization of Neurons Derived from ciNCCs

[0231] To determine the function of neurons derived from ciNCCs, we examined their electrophysiological properties. After co-culturing with rat cortical neurons for 10 - 20 days under differentiation conditions, whole-cell patch-clamp recordings were obtained from neurons derived from ciNCCs. The cultured neurons were transferred to a perfusion chamber on an Olympus BX51WI upright microscope and perfused at room temperature with artificial cerebrospinal fluid (aCSF) containing (in mM): NaCl 119, KCl 2.5, NaH2PO4 1, NaHCO3 26.2, and glucose 11, CaCl2 2.5, and MgSO4 1.3, and the osmolarity was adjusted to 300 osm L -1。During the entire recording process, aCSF was bubbled with 95% O2 and 5% CO2. Data were collected by a MultiClamp 700B amplifier (Axon Instruments), filtered at 2 kHz, and digitized at 10 kHz. Offline analysis was performed in Igor Pro (Wavemetrics). Action potentials were recorded in the current-clamp whole-cell configuration. The electrode solution for current-clamp experiments contained the following (in mM): potassium gluconate 123, KCl 10, MgCl2 1, HEPES 10, EGTA 1, CaCl2 0.1, K2ATP 1, Na4GTP 0.2, and glucose 4, and the pH was adjusted to 7.2 with KOH. The membrane potential was held at approximately -70 mV, and step currents from -20 to 50 pA were injected at 10-pA intervals. Whole-cell currents were recorded at a holding potential of -70 mV, and voltage steps in the range from -70 mV to +30 mV were delivered in 20-mV increments. Spontaneous postsynaptic currents were recorded in the whole-cell voltage-clamp mode. The whole-cell electrode solution for synaptic current recording contained the following (in mM): CsCl 135, HEPES 10, EGTA 1, Mg-ATP 4, Na4GTP 0.4, and QX-314 10, pH 7.4. To sample excitatory and inhibitory currents, 1 mM glutamate and 100 μM GABA were puffed for 100 ms at 10 p.s.i., and the holding voltages were -70 mV and 0 mV, respectively. As expected, it generated a repetitive sequence of action potentials evoked by depolarizing the membrane in the current-clamp mode, indicating that ciNCC source neurons had normal neuronal activity (data not shown).

[0232] 4. Cell proliferation assay

[0233] To measure the proliferative capacity of ciCECs, a cell proliferation assay was performed. The proliferation rate of ciCECs cultured in stage II medium was determined using the Click-iTTM EdU Alexa Fluor 488 Imaging Kit (Invitrogen / Life Technologies) according to the manufacturer's instructions. Briefly, passaged CECs were seeded at a low density of 5×10 3 cells / cm2 onto FNC-coated slides and cultured for 24 hours. The results showed that these cells had a high proliferative capacity (data not shown).

[0234] 5. Safety of ciCECs verified by teratoma formation assay

[0235] To evaluate the potential risk of tumorigenesis, 1×10 6The ciCECs were subcutaneously injected into NOD-SCID mice, and teratomas formed within 4 to 8 weeks. To generate chimeras, the ciCECs were injected into ICR blastocysts and transplanted into pseudopregnant ICR females. Germline transmission of the resulting chimeric mice was determined by mating the F2 mice with ICR mice. All animal experiments were approved by the Animal Care and Use Committee of the Guangzhou Institutes of Biomedicine and Health and were conducted in accordance with its guidelines. No tumors formed within 6 months after transplantation with ciCECs, while large teratomas appeared 4 - 8 weeks after transplantation in recipients transplanted with mESCs (data not shown). This indicates that ciCECs have minimal, if any, tumorigenic potential. In addition, ciCECs maintained a normal karyotype during 30 consecutive passages in vitro (data not shown). To better understand in vivo differentiation, ciCECs were transplanted into the eyes of NOD / SCID mice. After 4 - 8 weeks, no tumors formed from the transplanted ciCECs within 6 months after transplantation with ciCECs (data not shown).

[0236] 6. Transplantation of ciCECs into damaged eyes of rabbits

[0237] To assess whether ciCECs have the ability to transplant and expand in vivo, we transplanted them into a rabbit model of bullous keratopathy induced by mechanical scraping of the corneal endothelium from Descemet's membrane. The rabbits were divided into 2 groups (n = 10 per group), and the right eyes were used for this experiment. The ciCECs were injected into the anterior chamber ( Figure 6 Panel B, Image 1). Each recipient received 1×10 6 cells (low dose) of ciCECs or 2×10 6 cells (high dose) of ciCECs. Untreated normal rabbits ( Figure 6 Panel B, Image 4) and rabbits injected only with PBS ( Figure 6 Panel B, Image 3) were used as controls.

[0238] After disinfecting and aseptically covering the operative site, a 6-mm sclerocorneal incision centered at 12 o'clock was made with a crescent knife (Alcon Surgical, Shanghai, China), and viscoelastic agent (Healon; Amersham Pharmacia Biotech AB) was infused into the anterior chamber. After marking the corneal surface with a marker pen (Devon Industries, Madrid, Spain), a 6.0-mm diameter circular opening for Descemet's membrane removal (descemetorhexis) was created at the corneal center with a 30-gauge needle (Terumo, Tokyo, Japan), and Descemet's membrane was removed from the anterior chamber of the eye. The corneal endothelium was mechanically scraped from Descemet's membrane with a lacrimal duct irrigator (Weigao, Shandong). The Fsp - ciCEC was dissociated using 0.25% trypsin-EDTA and resuspended in PBS at a density of 1 × 10 7 cells / ml and kept on ice. The anterior chamber was washed three times with PBS.

[0239] After this procedure, a 26-gauge needle was used to inject 1 × 10 6 ciCEC suspended in 100 μl of basal DMEM containing 100 μM ROCK inhibitor Y-27632 (which promotes cell adhesion to the implantation site) (ROCK inhibitor, Selleck) into the anterior chamber of the right eye ( Figure 6 B, first image).

[0240] After the procedure, the rabbits were placed in the prone position for 2 - 3 hours. Each operative eye was examined externally two or three times a week and photographed at 1, 3, 5, 7, 14, 21, 35, and 42 days after surgery. Slit-lamp photographs showed a significant improvement in corneal transparency in the CEC-like cell group (ciCEC group) after injection, and the pupil and iris texture were visible. Only about 7 days later, the cornea became significantly transparent, while corneal opacity and stromal edema remained severe in the control group ( Figure 6 B, first and third images from the left). Visante OCT also showed a rapid decrease in corneal thickness after injection of CEC-like cells ( Figure 6 C, each image corresponding to the image above Figure 6 B). Confocal microscopy images confirmed complete coverage of polygonal cells on Descemet's membrane in the ciCEC group ( Figure 6 D, first image. Figure 6 Each image in D corresponds to the images above Figure 6 B and Figure 6 C). The mean corneal thickness at 1, 3, 5, 7, 14, 21, 35, and 42 days in the ciCEC group was found to be significantly less than that in the untreated control group ( Figure 6E). The slit lamp photographs showed a significant improvement in corneal transparency in the ciCEC group at days 1, 3, 7, 14, 21, and 28 ( Figure 7 ).

[0241] Example 4: Generation of ciNCC and ciCEC from fibroblasts according to another embodiment

[0242] Preparation of M6 Reprogramming Medium

[0243] The basal medium contained KnockOut DMEM (Gibco), 10% KSR (Gibco), 10% FBS (Gibco), 1% NEAA (Gibco), and 0.1 mM 2-mercaptoethanol (Gibco), and was supplemented with small molecules Chir99021 (3 μM), SB431542 (5 μM), forskolin (10 μM), VPA (500 mM), EPZ004777 (5 μM), and 5-aza-dC (0.5 μM). The medium was shaken for 30 minutes to ensure complete dissolution of all components.

[0244] Preparation of Differentiation Medium

[0245] DMEM / F12 / GlutaMAX (Gibco), 10% KSR (Gibco), 1% NEAA (Gibco), and 0.1 mM 2-mercaptoethanol (Gibco) were supplemented with SB431542 (5 μM) and CKI-7 (5 μM).

[0246] Chemical Conversion of Fibroblasts into NCC

[0247] MEFs were seeded at 5×10 4 cells / well in fibroblast medium in a 6-well tissue culture plate. The plates were pre-coated with fibronectin or laminin for over two hours. After overnight culture, the medium was replaced with M6 chemical medium, which was renewed every 2 days. NCC-like cells appeared and increased at days 3 - 5. After induction for 7 - 10 days, FACS sorting was performed to collect Wnt1 + cells.

[0248] Chemical Induction of CEC-Like Cells from Mouse ciNCC

[0249] At days 12 - 16, the M6 chemical medium was replaced with SB431542 and CKI-7 medium, which was renewed every 2 days. Endothelial-like cell clusters appeared and increased at day 8, and Oct4-GFP positive clusters appeared at day 12. CEC-like cells appeared as early as day 20. During days 30 to 35, CEC-like cell colonies were counted or further assayed.

[0250] Differentiation of ciNCC

[0251] Approximately 5×10 3 ciNCCs were seeded onto lathyronine-coated glass coverslips in a 24-well culture plate containing NCSC medium for the first 24 hours. After 24 hours, the cells were subjected to differentiation conditions. For neuronal differentiation, the medium was switched to neuronal differentiation medium (NCC medium without bFGF and EGF, supplemented with 200 μM ascorbic acid, 2 μM db-cAMP, 25 ng / ml BDNF, 25 ng / ml NT3, and 50 ng / ml GDNF). Half of the medium was changed every 2 - 3 days. Specific neuronal markers were analyzed from day 10 to day 20 after differentiation. For oligodendrocyte differentiation, the cells were cultured for 1 day in the presence of 5 μM retinoic acid and 200 ng / ml Shh and then for 3 - 5 days in the presence of 20 ng / ml PDGF-AA, 20 ng / ml bFGF, and 200 ng / ml SHH; subsequently, they were cultured in differentiation medium containing 40 ng / ml T3, 200 ng / ml Shh, 1 nM LDN193189, 5 mM db-cAMP, and 10 ng / ml NT3 for 8 - 12 days. The medium was renewed every other day. For astrocyte differentiation, 50 ng / ml BMP4 was added to the differentiation medium for 8 - 12 days, and the medium was changed every other day.

[0252] Results

[0253] Given that corneal endothelium is derived from NCCs, we designed a two-step method to reprogram mouse fibroblasts into CEC-like cells using small molecules. The first step was to direct the chemical reprogramming of mouse embryonic fibroblasts (MEFs) into ciNCCs. To screen for small molecules with the potential to convert fibroblasts into ciNCCs, we performed lineage tracing experiments to track the conversion process and exclude any NCCs or progenitor cells from the starting MEFs ( Figure 8 A, Figure 15 A). The Wnt1-Cre transgenic mouse has been established as a lineage tracing reporter model for NC development. In Wnt1-Cre / ROSA26tdTomato mice, the tdTomato protein is faithfully expressed in NCCs. Therefore, MEFs were isolated from Wnt1-Cre / ROSA26tdTomato mice at E13.5. Since the NCC population is labeled with tdTomato, we performed fluorescence-activated cell sorting (FACS) to collect the tdTomato-population to exclude any NCCs or progenitor cells (the purified cells are hereinafter referred to as Wnt1-tdTomato - MEFs; Figure 15B). We confirmed that Wnt1-tdTomato - MEFs were also negative for other NCC markers, including Sox10, P75, Pax3, Hnk1, and AP2α ( Figure 15 C, D). Additionally, these Wnt1-tdTomato - MEFs were negative for typical NSC markers, including Sox2, Pax6, and nestin (data not shown).

[0254] It has been reported that some small molecules used to enhance reprogramming can promote lineage reprogramming. To generate ciNCCs from MEFs, based on (1) epigenetic regulation and signaling regulation of NCC development and (2) enhanced neural lineage reprogramming, we selected a group of 16 small molecules as candidates. Initially, small molecule candidates for NC lineage reprogramming successfully focused on three classes of reprogramming factors, including Chir99021 (a GSK3 inhibitor), SB431542 (a TGF-β inhibitor), and forskolin (a cAMP agonist) ( Figure 8 B). For subsequent screening and optimization, we found that VPA (an HDAC inhibitor), EPZ004777 (a DOT1L inhibitor), and 5-aza-dC (a DNA methylation inhibitor) further enhanced the induction of Wnt1-tdTomato + cells ( Figure 8 C). In this study, we used a combination of chemically defined medium and the mixture of the above six small molecules (hereinafter referred to as M6) to reprogram MEFs into ciNCCs ( Figure 8 D). In the case of treatment with M6 medium, the expression of Wnt1-tdTomato was observed in individual cells as early as day 3 ( Figure 8 E). The M6 reprogramming medium effectively induced Wnt1-tdTomato + cells at 3.97% ( Figure 8 F). At days 5 - 7, induced Wnt1-tdTomato + colonies were observed in the M6 reprogramming medium ( Figure 8 G, Figure 15 E). These Wnt1-tdTomato + cells and colonies had a typical morphology similar to that of primary NCCs (pNCCs). At approximately day 12, the number of Wnt1-tdTomato + cells in small colonies increased significantly ( Figure 8 H). Although Wnt1-tdTomato +The efficiency of NCC generation was only the same as that of using TFs31 to transform human fibroblasts, but only 2 - 5% of the cells were Wnt1-tdTomato positive in this study. These results were reproducible in different batches of MEF (n = 8), and MEF with different genetic backgrounds (C57BL / 6, 129×C57BL / 6, and 129) could also be transformed into ciNCC by M6 condition. Collectively, these results indicate that M6 can reprogram MEF into ciNCC.

[0255] Example 5: Characterization of ciNCC and ciCEC obtained in Example 4 and additional studies

[0256] Characterization of Transformed ciNCC

[0257] The reprogramming process of ciNCC has two stages: the initial stage (days 0 - 7) and the expansion stage (days 7 - 12). The first stage is to culture MEF in the reprogramming medium to initiate epigenetic activation. A few small NCC-like clusters with clear edges appear. The second stage is to culture the epigenetically activated cells in the defined small molecule medium. Most of the clusters expand and grow gradually in the second stage.

[0258] To obtain ciNCC, we performed FACS to collect Wnt1-tdTomato + cells. The formed ciNCC were continuously propagated in the conventional NCC expansion medium containing N2, B27, bFGF, and EGF. Morphologically, the M6-induced cells at P3 maintained typical NCC characteristics in monolayer culture ( Figure 9 A). After passage, the ciNCC became morphologically uniform. To further characterize the M6-induced Wnt1-tdTomato + cells, we attempted to examine their gene expression. Our results showed that ciNCC expressed multiple NCC markers, including P75, HNK1, AP2α, and nestin ( Figure 9 B). In addition, we tested whether ciNCC had the differentiation potential towards peripheral neurons, Schwann cells, and others. For the differentiation of ciNCC, these cells were cultured in different lineage differentiation media. After 2 - 4 weeks of culture, the expression of markers was evaluated by immunostaining to examine the differentiated cells. Notably, ciNCC could also generate cells expressing specific markers of neurons (including Tuj1 and peripherin) ( Figure 9 C). For melanocyte differentiation, we observed melanocytes after 2 - 3 weeks of induction ( Figure 9 C). Our immunostaining results showed that ciNCC could differentiate into Schwann cells. The induced Schwann cells were GFAP+ and S100β+ cells ( Figure 9D). Further in vitro differentiation of these ciNCCs gave rise to mesenchymal lineages, resulting in typical mesenchymal cell morphology. Our results demonstrated that these ciNCC-derived mesenchymal cells could generate osteoblasts, adipocytes, and chondrocytes. Figure 9 E). Collectively, these data indicate that our ciNCCs can be induced to differentiate towards peripheral nervous system lineages and mesenchymal lineages.

[0259] Demonstration of ciCEC Function after Transplantation into Animal Models

[0260] To evaluate whether ciCECs have the ability to regenerate corneal endothelium in vivo, we transplanted them into a well-established rabbit model that had bullous keratopathy by mechanically scraping the corneal endothelium from Descemet's membrane. Previous studies found that injection of human pCECs supplemented with a ROCK inhibitor restored endothelial function. Based on this study, ciCECs were injected into the anterior chamber of the eye in combination with a ROCK inhibitor (Y27636). Figure 14 A). Each recipient received 1×10 6 ciCECs. The contralateral eye (normal) and the untreated eye (PBS injection) were used as experimental controls. Compared with the untreated eye, corneal edema decreased much earlier after ciCEC transplantation. Compared with the untreated eye that showed no change in corneal transparency, we noticed that the transparency of the cornea of the transplanted eye gradually increased after transplantation. Figure 14 B, Figure 19 A). After 7 days, the cornea of the transplanted eye became transparent, while corneal opacity and stromal edema remained poor in the untreated eye. Slit-lamp examination results also showed a significant improvement in the transparency of the cornea of the transplanted eye after injection, and the pupil and iris texture could be clearly observed. Figure 14 C, Figure 19 A).

[0261] Next, we investigated the survival of ciCECs in the transplanted eyes. The eyeballs were enucleated on the 28th day after surgery to evaluate the transplanted ciCEC cells. Fluorescence microscopy examination confirmed the presence of tdTomato-labeled transplanted cells. Immunohistochemistry showed ZO-1 expression, indicating the pump function of the transplanted ciCEC cells. Figure 14 D, Figure 19 C). Visante optical coherence tomography (OCT) of the anterior segment of the eye also showed a decrease in corneal thickness after ciCEC injection. Figure 14 E). Confocal microscopy confirmed complete coverage of polygonal endothelial cells on Descemet's membrane in the disease model with ciCEC transplantation, which could not be detected in the untreated model due to its severe corneal opacity. Figure 14G). Under magnification, at day 28, the transplanted ciCECs adhered tightly to the posterior corneal surface as a monolayer, while Descemet's membrane in the untreated model was exposed and had no detectable CECs.

[0262] There was a rapid decrease in corneal thickness within 4 weeks after ciCEC injection, followed by a more gradual decrease within the next 2 weeks ( Figure 14 F, Figure 19 D). In the untreated group, the average corneal thickness was approximately 1200 μm throughout the 42-day observation period. In contrast, it decreased rapidly in the transplanted group and was significantly less than the average corneal thickness in the untreated group. We observed that at days 14 (P < 0.01), 21, 28, 35, and 42 (P < 0.001) after surgery, the average corneal thickness in the ciCEC-transplanted group was significantly less than that in the control group, indicating a significant reversal of corneal edema. These results strongly suggest that ciCEC transplantation repopulates and self-organizes on the posterior corneal surface and has the ability to regenerate corneal endothelium.

[0263] Small Molecules Promote Induction of ciCEC from ciNCC

[0264] To generate mouse CEC-like cells from ciNCCs, we also sought a set of small molecules as candidates based on the importance of small molecules in in vitro CEC organogenesis and maintenance. In a preliminary screen, we found that SB431542 and CKI-7 were able to induce CEC-like cells from ciNCCs. These two compounds were subsequently included in the differentiation conditions ( Figure 10 A). To determine whether ciNCCs could be further differentiated into mature CECs, we treated ciNCCs with a differentiation medium containing these two small molecules. After culturing in this differentiation medium for 7 - 15 days, small populations outside or within the clusters showed typical tight aggregates with a uniform and polygonal morphology ( Figure 10 B). We also observed that these colonies expanded rapidly, and by days 12 - 15, small clusters merged into larger clusters ( Figure 10 B). These CEC-like cells grew rapidly and had strong proliferative capacity. The ciNCC-induced CEC-like cells formed a monolayer of hexagonal and pentagonal cells. To confirm that the CEC-like cells were derived from ciNCCs, we differentiated tdTomato + ciNCCs with a CEC differentiation medium containing 5 μM SB431542 and 5 μM CKI-7. These tdTomato + ciNCCs could then also be induced to differentiate towards CEC-like cells ( Figure 10 C). Na + / K +The expressions of -ATPase, AQP1, vimentin, ZO-1, and N-cadherin were further verified by immunofluorescence staining ( Figure 10 D). In this study, we identified the functions of CEC-like cells by the uptake of Dil-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) ( Figure 10 E). Global gene expression analysis by RNA sequencing showed that CEC-like cells shared a similar gene expression profile with primary CECs (pCECs), but this profile was different from that of naïve MEFs ( Figure 10 F). Tight junctions were observed in CEC-like cells by TEM ( Figure 10 G). To further monitor the reprogramming process, we further confirmed the expressions of a set of NCC and CEC markers at different times by using qRT-PCR. By day 12, robust expression of NCC genes, including Hnk1, P75, Sox10, Sox9, Pax3, and Ap2, was detected in the cells ( Figure 11 A). In addition, similar kinetics of gene activation of CEC genes (such as Slc4a1c, Col8a1, Na + / K + -ATPase, Aqp1, and N-cadherin) were also detected by qRT-PCR ( Figure 11 B). Genes known to be enriched in pCECs were significantly upregulated in ciCECs. To verify the conversion process from fibroblasts to CECs, we analyzed the transcriptome by using RNA sequencing ( Figure 11 C). The expressions of 16 CEC characteristic genes in ciCECs were significantly upregulated, consistent with pCECs. However, fibroblast characteristic genes were significantly downregulated. Notably, during the induction process, a set of NCC marker genes were initially upregulated and then downregulated. Principal component analysis showed that M6-treated cells were different from naïve MEFs, indicating that chemical reprogramming caused significant transcriptional changes ( Figure 11 D). These results indicated that CEC-like cells acquired CEC properties. Collectively, these data showed that the combination of SB431542 and CKI-7 effectively promoted the generation of CECs within 10 - 15 days in ciNCC cultures. Those CEC-like cells originated from fibroblasts were subsequently called chemically induced CEC-like cells (ciCECs).

[0265] Lineage Tracing to Confirm Induction of ciCEC from Fibroblasts

[0266] To confirm the origin of the initial fibroblasts used for small molecule-based reprogramming, we sought a genetic lineage tracing strategy to purify fibroblast-specific protein 1 (Fsp1)-tdTomato-positive fibroblasts ( Figure 12A). Fsp1-Cre has been validated as a specific fibroblast marker for lineage tracing; thus, Fsp1-Cre mice were crossed with ROSA26tdTomato mice. MEFs were isolated from transgenic mice (Fsp1-Cre / ROSA26tdTomato) at E13.5, and fibroblasts specifically expressed tdTomato; these cells were named tdMEFs hereinafter ( Figure 16 A). To avoid potential contamination of MEFs by NCC progenitors, we performed FACS to collect tdTomato + / p75 - populations ( Figure 12 B). These tdMEFs were negative for all NCC markers, including P75, HNK1, Sox10, and AP2α ( Figure 12 C).

[0267] Subsequently, these tdMEFs were induced with the above M6 medium. Epithelial clusters expressing tdTomato were observed during ciNCC induction ( Figure 12 D, Figure 16 B). We passaged Fsp1-tdTomato + ciNCCs and cultured them in NCC expansion medium for further experiments (after 2 weeks of induction). Immunofluorescence analysis confirmed that these Fsp1-tdTomato + ciNCCs were positive for NC markers P75, HNK1, SOX10, and AP2α, indicating that colonies of Fsp1-tdTomato + ciNCCs had differentiated towards CEC-like cells ( Figure 16 C). In addition, Fsp1-tdTomato + ciNCCs could differentiate into tdTomato + ciCECs ( Figure 16 D). By immunostaining, we found that the differentiated CEC-like cells co-expressed Na + / K + -ATPase, AQP1, laminin, ZO-1, Na + / K + -ATPase, and tdTomato ( Figure 12 e). Notably, all of these ciCECs also expressed tdTomato, demonstrating the conversion from fibroblasts. These results clearly confirmed that ciNCCs and ciCECs were converted from fibroblasts through a two-step lineage reprogramming.

[0268] ciCEC Generated by Chemicals Bypass the Induced Pluripotent Stem Cell (iPSC) Stage

[0269] Because the mechanism of our lineage reprogramming may be similar to that of chemically reprogrammed iPSCs, we sought to determine whether ciCECs undergo an iPSC stage. We performed a comparison between chemical iPSC reprogramming from MEFs, which were derived from mice carrying an Oct4 promoter-driven GFP (OG2) reporter, and ciCEC induction. We observed that these MEFs treated with M6 underwent a characteristic mesenchymal-to-epithelial transition (MET) morphologically, and small cell colonies gradually emerged near day 6 ( Figure 17 A). These cell colonies expressed the NCC marker Sox10 ( Figure 17 B). In contrast, we did not observe any Oct4-GFP-positive cells during the entire process from MEFs to ciCECs based on our method ( Figure 17 C, 3D). In addition, ciCECs maintained a normal karyotype during ten consecutive passages in vitro ( Figure 17 E). To evaluate the potential risk of tumorigenesis, a total of 5×10 6 ciCECs and 2×10 6 mouse embryonic stem cells (ESCs) were subcutaneously transplanted into NOD / SCID mice. Notably, no tumors formed within 6 months after transplantation with ciCECs, while large teratomas appeared in mice transplanted with ESCs after 4 - 8 weeks (data not shown). This result indicates that ciCECs do not have tumorigenic potential. To better understand their in vivo differentiation, we transplanted ciCECs into the anterior chamber of the eye of NOD / SCID mice. After 4 - 8 weeks and within 6 months after transplantation, the transplanted ciCECs did not form tumors. These results demonstrate that our method can directly reprogram MEFs into ciNCCs and ultimately into ciCECs, bypassing the iPSC stage.

[0270] Long-Term In Vitro Expansion Ability of ciCEC

[0271] It has been demonstrated that it is challenging to maintain the morphology and normal physiological functions of CECs in vitro. We aimed to test whether a large number of functional ciCECs could be generated from fibroblasts to enable large-scale application of ciCECs. Based on our observation that ciCECs cultured in medium containing SB431542 (5 μM) and CKI-7 (5 μM) are small hexagonal cells (without epithelial-to-mesenchymal transition-like cells) ( Figure 18 A), we hypothesized that SB431542 and CKI-7 would promote ciCEC growth in vitro. We evaluated the long-term in vitro expansion ability of ciCECs by serially passaging ciCECs at a 1:6 ratio and found that the phenotype was similar between P3 and P30 ( Figure 13A, B). This result shows that SB431542 and CKI-7 strongly promote the expansion of ciCECs. In the small molecule-based medium, these ciCECs maintain themselves as a homogeneous cell population with a hexagonal morphology for at least 30 passages (P30). Additionally, we successfully cloned and cultured these ciCECs to 10 passages and demonstrated consistent morphology. Our immunostaining results show that the ratio of Ki67-positive cells in ciCECs at P3 is higher than that in pCECs at P3( Figure 18 B). ciCECs are highly proliferative because 24.6%, 37.8%, and 48.1% of these cells at P1, P3, and P6 showed EdU incorporation( Figure 18 C). The results of FACS analysis using propidium iodide (PI) staining show that the cell cycle distribution (G0 / G1, S, and G2 / M phases) is 46.30%, 45.11%, and 8.59% for ciCECs at P3 and 67.30%, 21.50%, and 11.20% for pCECs at P3( Figure 18 D). They rapidly expand into large homogeneous colonies with a population doubling time of 22.3 ± 3.7 hours( Figure 13 C). Notably, large "vacuole-like" structures were found on the surface of ciCECs at P2 to P10( Figure 13 D). These vacuole-like structures disappeared at P20 when the cells were continuously propagated. Notably, ciCECs at P30 also expressed typical CEC markers, including Na + / K + -ATPase, AQP1, and ZO-1( Figure 13 E). When analyzing the ability to migrate into the gap created by a scratch through imaging, ciCECs at different passage numbers cultured in the medium containing SB431542 and CKI-7 showed stronger proliferation and migration abilities compared to pCECs( Figure 18 E, F). Collectively, these results demonstrate that SB431542 and CKI-7 have a robust and universal effect on the long-term expansion of ciCECs in vitro.

Claims

1. A method for reprogramming a first type of cell into a third type of cell, comprising the steps of: (a) culturing the first type of cell in the presence of a first set of reprogramming factors, wherein the first set of reprogramming factors comprises a glycogen synthase kinase 3 inhibitor, a transforming growth factor-β inhibitor, and a cyclic AMP inducer; and (b) culturing the neural crest cell-like cells obtained from step (a) in the presence of a second set of reprogramming factors, wherein the second set of reprogramming factors comprises a transforming growth factor-β inhibitor and a casein kinase 1 inhibitor; wherein the first type of cell is a fibroblast, a human umbilical cord mesenchymal stromal cell, or a urine cell, and the third type of cell is a corneal endothelial cell-like cell; The fibroblasts are selected from the group consisting of: mouse embryonic fibroblasts (MEF), mouse tail tip fibroblasts (TTF), human embryonic fibroblasts (HEF), human neonatal fibroblasts (HNF), adult fibroblasts (HAF), human foreskin fibroblasts (HFF), and mixtures thereof; The glycogen synthase kinase 3 inhibitors are selected from the group consisting of: CHIR99021, LiCl, Li2CO3, BIO ((2’Z,3’E)-6-bromoindirubin-3’-oxime), TD114-2, camphorquinone, TWS119, CBM1078, SB216763, 3F8 (TOCRIS), AR-A014418, FRATide, indirubin 3’-oxime, and L803; Wherein the transforming growth factor-β inhibitors are selected from the group consisting of: SB431542, Repsox, 616452, LDN193189, A8301, GW788388, SD208, SB525334, LY364947, D4476, SB505124, and tranilast. The cyclic AMP inducers are forskolin, IBMX, rolipram, 8BrcAMP, prostaglandin E2, NKH477, dibutyryl-cyclic adenosine monophosphate, Sp-8-Br-cAMPs; The casein kinase 1 inhibitor is CKI-7; The concentration of the glycogen synthase kinase 3 inhibitor is 0.1 - 10 μM; The concentration of the cyclic AMP inducer is 1 - 20 μM; The concentration of the transforming growth factor-β inhibitor is 0.1 - 20 μM; The concentration of the casein kinase 1 inhibitor is 0.1 - 20 μM.

2. The method according to claim 1, wherein the first set of reprogramming factors further comprises basic fibroblast growth factor, a DNA methyltransferase inhibitor, a DOT1L inhibitor, a histone deacetylase inhibitor, BMP4, or a combination thereof.

3. The method according to claim 1, wherein the first set of reprogramming factors consists of: (a) A glycogen synthase kinase 3 inhibitor, a transforming growth factor-β inhibitor, and a cyclic AMP inducer, (b) a glycogen synthase kinase 3 inhibitor, a transforming growth factor-β inhibitor, a cyclic AMP inducer, and a basic fibroblast growth factor, or (c) a glycogen synthase kinase 3 inhibitor, a transforming growth factor-β inhibitor, a cyclic AMP inducer, a DNA methyltransferase inhibitor, a DOT1L inhibitor, and a histone deacetylase inhibitor.

4. The method according to claim 2, wherein the DNA methyltransferase inhibitor is selected from the group consisting of 5-aza-dC, 5-azacytidine, and RG108.

5. The method according to claim 2, wherein the DOT1L inhibitor is EPZ004777.

6. The method according to claim 2, wherein the histone deacetylase inhibitor is selected from the group consisting of valproic acid, trichostatin A, vorinostat, apicidin, trapoxin, depudecin, FR901228, and butyrate.

7. The method according to claim 1, wherein the neural crest cell-like cells are positive for P75, Hnk1, AP2α, and Sox10.

8. The method according to claim 1, wherein the first type of cells is cultured in the presence of the first set of reprogramming factors for (a) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days or (b) not more than 20, 19, 18, 17, 16, 15, 14, 13, or 12 days.

9. The method according to claim 1, the method further comprising washing the cells obtained from step (a) before starting step (b).

10. The method according to claim 1, wherein there is no washing step between step (a) and step (b).

11. The method according to claim 1, wherein the second set of reprogramming factors further comprises BMP4 and / or a DNA methyltransferase inhibitor.

12. The method according to claim 11, wherein the DNA methyltransferase inhibitor is selected from the group consisting of 5-aza-dC, 5-azacytidine, and RG108.

13. The method according to claim 1, wherein the corneal endothelial cell-like cells are positive for ZO-1 and Na + / K + -ATPase.

14. The method according to claim 1, wherein the cells obtained from step (a) are cultured in the presence of the second set of reprogramming factors for (a) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days or (b) not more than 20, 19, 18, 17, 16, 15, 14, 13, or 12 days.

15. A kit for reprogramming a first type of cell into a third type of cell, wherein the kit comprises a first set of reprogramming factors and a second set of reprogramming factors, wherein the first set of reprogramming factors comprises a glycogen synthase kinase 3 inhibitor, a transforming growth factor-β inhibitor, and a cyclic AMP inducer, and the second set of reprogramming factors comprises a transforming growth factor-β inhibitor and a casein kinase 1 inhibitor; wherein the first type of cell is a fibroblast, a human umbilical cord mesenchymal stromal cell, or a urine cell, and the third type of cell is a corneal endothelial cell-like cell; wherein the transforming growth factor-β inhibitor is selected from the group consisting of: SB431542, Repsox, 616452, LDN193189, A8301, GW788388, SD208, SB525334, LY364947, D4476, SB505124, and tranilast. The cyclic AMP inducer is forskolin, IBMX, rolipram, 8BrcAMP, prostaglandin E2, NKH477, dibutyryl-cyclic adenosine monophosphate, Sp-8-Br-cAMPs; The casein kinase 1 inhibitor is CKI-7; The concentration of the glycogen synthase kinase 3 inhibitor is 0.1 - 10 μM; The concentration of the cyclic AMP inducer is 1 - 20 μM; The concentration of the transforming growth factor-β inhibitor is 0.1 - 20 μM; The concentration of the casein kinase 1 inhibitor is 0.1 - 20 μM.

16. The kit according to claim 15, wherein the first set of reprogramming factors further comprises a DNA methyltransferase inhibitor, a DOT1L inhibitor, and a histone deacetylase inhibitor.

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